Novel expression vectors and uses thereof

ABSTRACT

A method for treating an HIV disease in a subject in need of said treatment, comprising administering to the subject a therapeutically effective amount of a DNA vaccine comprising an expression vector and a pharmaceutically acceptable excipient, where the expression vector comprises: (a) a heterologous promoter operatively linked to a DNA sequence encoding a nuclear-anchoring protein, where the nuclear-anchoring protein comprises: (i) a DNA binding domain which binds to a specific DNA binding sequence, and (ii) a functional domain of the Bovine Papilloma Virus Type 1 E2 protein, where the functional domain binds to a nuclear component; (b) a multimerized DNA sequence that forms a binding site for the nuclear anchoring protein; and (c) at least one expression cassette comprising a DNA sequence encoding a protein or peptide that stimulates an immune response specific to the protein or peptide; where the expression vector lacks an origin of replication functional in mammalian cells.

1. FIELD OF THE INVENTION

The present invention relates to novel vectors, to DNA vaccines and genetherapeutics containing said vectors, to methods for the preparation ofthe vectors and DNA vaccines and gene therapeutics containing thevectors, and to therapeutic uses of said vectors. More specifically, thepresent invention relates to novel vectors comprising (a) an expressioncassette of a gene of a nuclear-anchoring protein, which contains (i) aDNA binding domain capable of binding to a specific DNA sequence and(ii) a functional domain capable of binding to a nuclear component and(b) a multimerized DNA forming a binding site for the anchoring proteinof a nuclear-anchoring protein, and optionally (c) one or moreexpression cassettes of a DNA sequence of interest. In particular theinvention relates to vectors that lack a papilloma virus origin ofreplication. The invention also relates to vectors that lack an originof replication functional in a mammalian cell. The invention furtherrelates to methods for expressing a DNA sequence of interest in asubject.

2. BACKGROUND OF THE INVENTION

Transfer of autologous or heterologous genes into animal or humanorganisms with suitable vectors is emerging as a technique with immensepotential to cure diseases with a genetic background or to prevent orcure infectious diseases. Several types of viral and non-viral vectorshave been developed and tested in animals and in human subjects todeliver a gene/genes that are defective by mutations and thereforenon-functional. Examples of such vectors include Adenovirus vectors,Herpes virus vectors, Retrovirus vectors, Lentivirus vectors andAdeno-associated vectors.

Vaccination has proven to be a highly effective and economical method toprevent a disease caused by infectious agents. Since the introduction ofthe Vaccinia virus as an attenuated vaccine against the smallpox virus(Variola), vaccines against a multitude of human pathogens have beendeveloped and taken into routine use. Today small pox has beeneradicated by vaccinations and the same is to be expected shortly forthe poliovirus. Several childhood diseases, such as pertussis,diphtheria and tetanus, can be effectively prevented by vaccinations.

In general, the most successful viral vaccines are live avirulentmutants of the disease-causing viruses. The key to the success of thisapproach is the fact that a living virus targets the same organs, thesame type and similar number of cells, and therefore, by multiplying inthe recipient, elicits a long-lasting immune response without causingthe disease or causing only a mild disease. In effect, a live attenuatedvaccine produces a subclinical infection, the nature's own way ofimmunizing. As a result, a full immune response will be induced,including humoral, cellular and innate responses, providing a longlasting and sometimes a life-long immune protection against thepathogen.

Although live attenuated vaccines are most potent, they can causeharmful side effects. Thus, an attenuated viral vaccine can revert to avirulent strain or in cases where the attenuated virus is apathogenic inadults it can still cause a disease in infants or in disabled persons.This is true in the case of viruses causing chronic infections, such asHuman Immunodeficiency Virus type 1 and 2. Vaccines composed of viraland bacterial proteins or immunogenic peptides are less likely to causeunwanted side effects but may not be as potent as the live vaccines.This is especially the case with vaccines against microbes causingchronic infections, such as certain viruses and intracellular bacteria.

The strength and type of immune response is, however, also dependent onhow the viral proteins are processed and how they are presented to theimmune system by antigen presenting cells (APCs), such as macrophagesand dendritic cells. Protein and peptide antigens are taken up by APCsvia endocytosis, processed to small immunogenic peptides through anendosomal pathway and presented to T-lymphocytes (T-cells) by MHC (majorhistocompatibility complex) class II antigens [in man HLAs (humanleukocyte antigens) class II]. In contrast, proteins synthesized de novoin APCs or in possible target cells for an immune response, will beprocessed through a cytoplasmic pathway and presented to T-cells by MHCclass I antigens (in man HLAs class 1). In general, the presentation ofimmunogenic peptides through the class II pathway will lead to theactivation of the helper/inducer T-cells, which in turn will lead to theactivation of B-cells and to antibody response. In contrast,presentation through class I MHC favors the induction of cytotoxicT-lymphocytes (CTLs), which are capable of recognition and destructionof virally infected cells.

In early 1990's, a method to mimic the antigen processing andpresentation that was normally achieved by live attenuated vaccines wasintroduced [Ulmer, J. B. et al Science 259 (1993) 1745-1749]. It wasshown that an injection of eukaryotic expression vectors in the form ofcircular DNA into the muscle induced take-up of this DNA by the musclecells (and probably others) and was able to induce the expression of thegene of interest, and to raise an immune response, especially a cellularimmune response in the form of CTLs, to the protein encoded by theinserted gene. Since that observation, DNA immunization has become astandard method to induce immune responses to foreign proteins inexperimental animals and human studies with several DNA vaccines areunderway.

Generally, the DNA vectors used in these vaccine studies contain acloning site for the gene of interest, a strong viral promoter, such asthe immediate early promoter of the CMV virus, in order to drive theexpression of the gene of interest, a polyadenylation region, and anantibiotic resistance gene and a bacterial replication origin for thepropagation of the DNA vector (plasmid) in bacterial cells.

With the vectors described above it is possible to obtain a detectablelevel of expression of the gene of interest after administering thevector to experimental animals or to humans, either by a directinjection to muscle or to skin with a particle bombardment technique orby applying the vector in a solution directly to mucous membranes.However, the expression obtained by these vectors is short lived: thevectors tend to disappear from the transfected cells little by littleand are not transferred to daughter cells in a dividing cell population.The short-term expression of the gene of interest and limited number ofcells targeted are probably the major reasons, why only temporary immuneresponses are observed in subjects immunized with DNA vectors describedabove. Thus, for example, Boyer et al. observed only temporary immuneresponses to HIV-1 Env and Rev proteins in human subjects, who wereimmunized several times with a vector similar to the those describedabove [Boyer, J. D., J Infect Dis 181 (2000) 476-483].

There is a growing interest in developing novel products useful in genetherapy and DNA vaccination. For instance papilloma virus vectorscarrying the expression cassette for the gene of interest have beensuggested to be useful candidates.

To date more than 70 subtypes of human papilloma viruses (HPVs) and manydifferent animal papilloma viruses have been identified [zur Hausen, H.and de Villiers E., Annu Rev Microbiol 48 (1994) 427-447; Bernard, H.,et al., Curr Top Microbiol Immunol 186 (1994) 33-54]. All papillomaviruses share a similar genome organization and the positioning of allof the translational open reading frames (ORFs) is highly conserved.

Papilloma viruses infect squamous epithelial cells of skin or mucosa atdifferent body sites and induce the formation of benign tumors, which insome cases can progress to malignancy. The papilloma virus genomes arereplicated and maintained in the infected cells as multicopy nuclearplasmids. The replication, episomal maintenance, expression of the lategenes and virus assembly are tightly coupled to the differentiation ofthe epithelial tissue: the papilloma virus DNA episomal replicationtakes place during the initial amplificational replication and thesecond, i.e. latent, and the third, i.e. vegetative, replications in thedifferentiating epithelium [Howley, P. M.; Papillomavirinae: the virusesand their replication. In Virology, Fields, B. C., Knipe, D. M., Howley,P. M., Eds., Lippincott-Raven Publishers, Philadelphia, USA, 1996, 2.Edition, p. 2045-2076].

Two viral factors encoded by the E1 and E2 open reading frames have beenshown to be necessary and sufficient for the initiation of the DNAreplication from the papilloma virus origin in the cells [Ustav, M. andStenlund, A., EMBO J 10 (1991) 449-57; Ustav, M., et al., EMBO J 10(1991) 4321-4329; Ustav, E., et al., Proc Natl Acad Sci USA 90 (1993)898-902].

Functional origins for the initiation of the DNA replication have beendefined for BPV1 [Ustav, M., et al., EMBO J 10 (1991) 4321-4329], HPV1a[Gopalakrishnan, V. and Khan, S., supra], HPV11 [Russell, J., Botchan,M., J Virol 69 (1995) 651-660], HPV18 [Sverdrup, F. and Khan, S., JVirol 69 (1995) 1319-1323: Sverdrup, F. and Khan, S., J Virol 68 (1994)505-509] and many others. Characteristically, all these origin fragmentshave a high A/T content, and they contain several overlapping individualE1 protein recognition sequences, which together constitute the E1binding site [Ustav, M., et al., EMBO J 10 (1991) 4321-4329; Holt, S.,et al., J Virol 68 (1994) 1094-1102; Holt, S, and Wilson, V., J Virol 69(1995) 6525-3652; Sedman, T., et al. J Virol 71 (1997) 2887-2996]. Inaddition, these functional origin fragments contain an E2 binding site,which is essential for the initiation of DNA replication in vivo in mostcases (Ustav, E., et al., supra). The E2 protein facilitates the firststep of the origin recognition by E1. After the initial binding ofmonomeric E1 to the origin the multimerization of E1 is initiated. Thisleads to the formation of the complex with the ori melting activity. Ithas been suggested that E2 has no influence on the following stages ofthe initiation of the DNA replication [Lusky, M., et al., Proc Natl AcadSci USA 91 (1994) 8895-8899].

The BPV1 E2 ORF encodes three proteins that originate from selectivepromoter usage and alternative mRNA splicing [Lambert, P., et al., AnnuRev Genet. 22 (1988) 235-258]. All these proteins can form homo- andheterodimers with each other and bind specifically to a 12 bpinterrupted palindromic sequence 5′-ACCNNNNNNGGT-3′ [Androphy, E., etal., Nature 325 (1987) 70-739].

There are 17 E2 binding sites in the BPV1 genome and up to four sites inthe HPV genomes, which play a crucial role in the initiation of viralDNA replication (Ustav, E., et al., supra) and in the regulation ofviral gene expression (Howley, P. M., Papillomavirinae: the viruses andtheir replication, in Virology, Fields, B. C., Knipe, D. M., Howley, P.M., Eds., Philadelphia: Lippincott-Raven Publishers, 1996. 2. edition,p. 2045-2076). Structural and mutational analyses have revealed threedistinct functional domains in the full size E2 protein. The N-terminalpart (residues 1 to 210) is an activation domain for transcription andreplication. It is followed by the unstructured hinge region (residues211 to 324) and the carboxy-terminal DNA binding-dimerization domain(residues 325 to 410) [Dostatni, N., et al., EMBO J 7 (1988) 3807-3816;Haugen, T., et al. EMBO J 7 (1988) 4245-4253; McBride, A., et al., EMBOJ 7 (1988) 533-539; McBride, A., et al., Proc Natl Acad Sci USA 86(1989) 510-514]. On the basis of X-ray crystallographical data, the DNAbinding-dimerization domain of E2 has a structure of a dyad-symmetriceight-stranded antiparallel beta barrel, made up of two identical“half-barrel” subunits [Hegde, R., et al., Nature 359 (1992) 505-512;Hegde, R., J Nucl Med 36 (6 Suppl) (1995) 25S-27S]. The functionalelements of the trans-activation domain of E2 have a very highstructural integrity as confirmed by mutational analysis [Abroi, A., etal., J Virol 70 (1996) 6169-6179; Brokaw, J., et al., J Virol 71 (1996)23-29; Grossel, M., et al., J Virol 70 (1996) 7264-7269; Ferguson, M.and Botchan, M., J Virol 70 (1996) 4193-4199] and by X-raycrystallography [Harris, S., and Botchan, M. R., Science 284 (1999)1673-1677 and Antson, A. et al., Nature 403 (2000) 805-809]. Inaddition, X-ray crystallography shows that the N-terminal domain of theE2 protein forms a dimeric structure, where Arg 37 has an importantfunction in dimer formation (Antson, A., et al., supra).

As has been described previously, bovine papillomavirus type 1 E2protein in trans and its multiple binding sites in cis are bothnecessary and sufficient for the chromatin attachment of the episomalgenetic elements. The phenomenon is suggested to provide a mechanism forpartitioning viral genome during viral infection in the dividing cells[Ilves, I., et al., J Virol. 73 (1999) 4404-4412].

None of the papilloma vectors or other vectors disclosed so far fulfillsthe criteria and requirements set forth for an optimal vaccine, whichare the same for DNA vaccines and for conventional vaccines. (It shouldbe noted that these requirements are preferred but not necessary for useas a vaccine.) First, an optimal vaccine must produce protectiveimmunity with minimal adverse effects. Thus the vaccine should be devoidof components, which are toxic and/or cause symptoms of the disease tothe recipient. Second, an optimal vaccine must induce apathogen-specific immune response, i.e. it must elicit a strong andmeasurable immune response to the desired pathogen without causing animmune response to other components of the vaccine. These tworequirements imply that a vector to be used as a DNA vaccine shouldoptimally only express the desired gene(s) and optimally should notreplicate in the host or contain any sequences homologous with those ofthe recipient, since nucleotide sequences that are homologous betweenthe vector and the host's genome may effect the integration of thevector into the host's genome. Third, an optimal vaccine must induce aright type of immune response; i.e. it must raise both humoral andcellular immune responses in order to act on the intracellular andextracellular pathogen. Finally, an optimal vaccine must be stable, i.e.it must retain its potency for a sufficiently long time in the body toraise the immune response in a vaccine formulation for use in variousdemanding circumstances during storage and preparation. Additionally,vaccines should be of reasonable price. Further, the route and themethod of inoculation are important considerations for optimizing a DNAimmunization.

When developing a DNA vaccine the stability of the expression of thedesired gene is sometimes a major problem. Thus, the maintenancefunction or the persistance of the vector in the recipient cell has beenfocused on in the prior art, however, often at the cost of the safety.For example, Ohe, Y., et al.][Hum Gene Ther 6 (3) (1995) 325-333]disclose a papilloma virus vector capable of stable, high-level geneexpression, which is suggested for use in gene therapy. Trans-formingearly genes E5, E6, and E7 have been deleted from said vector, but itstill contains nucleotide sequences encoding other papilloma viralgenes, such as the E1 and E2 genes, which are involved in thereplication of the virus. Thus, the vector produces several otherpapilloma proteins, which may elicit undesired immune responses andwhich induce a risk of the vector's integration in the recipient. Also,the vector is replicable, since it contains the E1 gene. Additionally,it is large in size and therefore subject to bacterial modificationduring preparation.

International Patent Application PCT/EE96/00004 (WO 97/24451) disclosesvectors capable of a long-term maintenance in a host cell and methodsusing such vectors for obtaining long-term production of a gene productof interest in a mammalian host cell, which expresses E1 and E2. Thesevectors contain a minimal origin of replication of a papilloma virus(MO), a Minichromosome Maintenance Element (MME) of a papilloma virusand a gene encoding said gene product, the MO and MME consisting of aDNA sequence different from the natural papilloma virus sequence, and insome embodiments the E1 gene. Additionally, vectors containing an MMEconsisting essentially of ten E2 binding sites are disclosed in someexamples. These vectors require the presence of the E1 protein either inthe host or in the vector for the expression. This imparts thereplication function to the vectors. These vectors also express the E1protein in addition to the gene of interest and the E2 protein andcontain sequences, such as rabbit β-globin sequences, which arepartially homologous to human sequences causing a serious risk ofintegration to human genome, which reduces the potential of thesevectors as DNA vaccines. Additionally, the vectors are unstable due totheir size (ca 15 kb): at the preparation stage in a bacterial cell, thebacterial replication machinery tends to modify the vector by randomslicing of the vector, which leads to unsatisfactory expression productsincluding products totally lacking the gene of interest.

International Patent Application PCT/EE96/00004 (WO 97/24451) furtherdiscloses that E1 and E2 are the only viral proteins necessary for theepisomal long-term replication of the vectors. Additionally, themaintenance function of the BPV1 genome is associated with the presenceof minimal ori (MO), which is stated to be necessary, although notsufficient, for the long-term persistence or the stable maintenance ofthe vectors the cells. In addition, the cis-elements, i.e. theMinichromosome Maintenance Elements of the BPV1, are stated to berequired for the stable replication of BPV1. In particular, multimericE2 binding sites (E2BS) are stated to be necessary for the stablemaintenance of the vectors.

There is a clear need for improved novel vectors, which would be usefulas DNA vaccines.

An object of the invention is therefore to provide novel vectors, whichare capable of a long-term maintenance in a large and increasing numberof different cells of the host's body and thereby capable of providing astable expression of the desired antigen(s).

Another object of the invention is to provide novel vectors, which aremaintained for a long period of time in the cells that originallyreceived the vector and transferred it to the daughter cells aftermitotic cell division.

Yet another object of the invention is to provide novel vectors, whichexpress in addition to the gene or genes of interest preferably only agene necessary for a long-term maintenance in the recipient cells andthus are devoid of components that are toxic or cause symptoms of thedisease to the recipient.

A further object of the invention is to provide novel vectors, whichmimic attenuated live viral vaccines, especially in their function ofmultiplying in the body, without inducing any considerable signs ofdisease and without expressing undesired proteins, which may induceadverse reactions in a host injected with the DNA vaccine.

Still a further object of the invention is to provide novel vectors,which do not replicate in the recipient.

Still another object of the invention is to provide novel vectors, whichinduce both humoral and cellular immune responses when used as DNAvaccines.

Yet another object of the invention is to provide novel vectors, whichare suitable for a large-scale production in bacterial cell.

Yet another object of the invention is to provide novel vectors, whichare not host specific and thus enable the production in variousbacterial cells.

An additional object of the invention is to provide novel vectors, whichare useful as carrier vectors for a gene or genes of interest,

A further object of the invention is to provide novel vectors, which areuseful in gene therapy and as gene therapeutic agents and for theproduction of macromolecular drugs in vivo.

3. SUMMARY OF THE INVENTION

The present invention discloses novel vectors, which meet therequirements of a carrier vector of a gene or genes of interest or of anoptimal DNA vaccination vector and which are preferably devoid ofdrawbacks and side effects of prior art vectors.

The present invention is based on the surprising finding that a vector(plasmid) carrying (i) an expression cassette of a DNA sequence encodinga nuclear-anchoring protein, and (ii) multiple copies of high affinitybinding sites for said nuclear-anchoring protein spreads inproliferating cells. As a result, the number of vector-carrying cellsincreases even without the replication of the vector. When the vectoradditionally carries a gene or genes of interest, the number of suchcells that express a gene or genes of interest similarly increaseswithout the replication of the vector. Thus, the vector of the inventionlacks a papilloma virus origin of replication. In a preferredembodiment, the vector of the invention lacks an origin of replicationthat functions in a mammalian cell.

Accordingly, the present invention discloses novel vectors useful ascarrier vectors of a gene or genes of interest, in DNA vaccination andgene therapy and as gene therapeutic agents. In a specific embodiment,said vectors are capable of spreading and, if desired, of expressing agene or genes of interest in an increasing number of cells for anextended time. The vectors of the present invention preferably expressonly a nuclear-anchoring protein, and, if desired, the gene or genes ofinterest, and optionally a selectable marker. However, they preferablylack any redundant, oncogenically transforming or potentially toxicsequences, thereby avoiding a severe drawback of the vectors previouslydisclosed or suggested for use as DNA vaccines, i.e. hypersensitivityreactions against other viral components. In certain embodiments of theinvention, this is achieved by low level of the expressednuclear-anchoring protein in the cells. At the same time, the vectors ofthe present invention induce both humoral and cellular immune responses,where the gene or genes of interest is included in the vector.

The vectors of the present invention are advantageous for use both invitro (e.g., in the production level) and in vivo (e.g., vaccination).

The present invention relates to the subject matter of the invention asset forth in the attached claims.

The present invention relates to expression vectors comprising: (a) aDNA sequence encoding a nuclear-anchoring protein operatively linked toa heterologous promoter, said nuclear-anchoring protein comprising (i) aDNA binding domain which binds to a specific DNA sequence, and (ii) afunctional domain that binds to a nuclear component, or a functionalequivalent thereof; and (b) a multimerized DNA sequence forming abinding site for the nuclear anchoring protein, wherein said vectorlacks a papilloma virus origin of replication. In a preferred embodimenta vector of the invention lacks an origin of replication functional in amammalian cell.

In certain embodiments, the nuclear component is mitotic chromatin, thenuclear matrix, nuclear domain 10 (ND10), or nuclear domain POD.

In certain specific embodiments, the nuclear anchoring-protein is achromatin-anchoring protein, and said functional domain binds mitoticchromatin.

In certain embodiments, the nuclear-anchoring protein contains a hingeor linker region.

In certain embodiments, the nuclear-anchoring protein is a naturalprotein of eukaryotic, prokaryotic, or viral origin. In certain specificembodiments, the natural protein is of viral origin.

In certain embodiments, the nuclear-anchoring protein is a naturalprotein of eukaryotic origin.

In certain embodiments, the nuclear-anchoring protein is that of apapilloma virus or an Epstein-Barr virus.

In specific embodiments, the nuclear-anchoring protein is the E2 proteinof Bovine Papilloma Virus type 1 or Epstein-Barr Virus Nuclear Antigen1.

In a specific embodiment, the nuclear-anchoring protein is the E2protein of Bovine Papilloma Virus type 1.

In specific embodiments, the nuclear-anchoring protein is a HighMobility Group protein.

In certain embodiments, the nuclear-anchoring protein is a non-naturalprotein.

In certain embodiments, the nuclear-anchoring protein is a recombinantprotein, a fusion protein, or a protein obtained by molecular modelingtechniques.

In specific embodiments, the recombinant protein, fusion protein, orprotein obtained by molecular modeling techniques contains anycombination of a DNA binding domain which binds to said specific DNAsequence and a functional domain which binds to a nuclear component,wherein said functional domain which binds to a nuclear component isthat of a papilloma virus, an Epstein-Barr-Virus, or a High MobilityGroup protein.

In certain specific embodiments, the recombinant protein, fusionprotein, or protein obtained by molecular modeling techniques containsany combination of a DNA binding domain which binds to said specific DNAsequence and a functional domain which binds to a nuclear component,wherein said functional domain which binds to a nuclear component isthat of E2 protein of Bovine Papilloma Virus type 1, Epstein-Barr VirusNuclear Antigen 1, or a High Mobility Group protein.

In certain embodiments, the vector further comprises one or moreexpression cassettes of a DNA sequence of interest.

In certain embodiments, the DNA sequence of interest is that of aninfectious pathogen. In certain embodiments, the infectious pathogen isa virus. In certain specific embodiments, the virus is selected from thegroup consisting of Human Immunodeficiency Virus (HIV), Herpex SimplexVirus (HSV), Hepatitis C Virus, Influenzae Virus, and Enterovirus.

In certain embodiments, the DNA sequence of interest is that of abacterium. In certain embodiments, the bacterium is selected from thegroup consisting of Chlamydia trachomatis, Mycobacterium tuberculosis,and Mycoplasma pneumonia. In a specific embodiment, the bacterium isSalmonella.

In certain embodiments, the DNA sequence of interest is that of a fungalpathogen. In certain embodiments, the fungal pathogen is Candidaalbigans.

In certain embodiments, the DNA sequence of interest is of HIV origin.

In specific embodiments, the DNA sequence of interest encodes anon-structural regulatory protein of HIV. In more specific embodiments,the non-structural regulatory protein of HIV is Nef, Tat and/or Rev. Ina specific embodiment, the non-structural regulatory protein of HIV isNef.

In certain embodiments, the DNA sequence of interest encodes astructural protein of HIV. In a specific embodiment, the DNA sequence ofinterest is the gene encoding HIV gp120/gp160.

In certain embodiments, the vector of the invention comprises a firstexpression cassette comprising a DNA sequence of interest which encodesNef, Tat and/or Rev, and a second expression cassette comprising a DNAsequence of interest which encodes Nef, Tat and/or Rev.

In certain embodiments, the vector of the invention comprises a firstexpression cassette comprising a DNA sequence of interest which encodesNef, Tat and/or Rev, and a second expression cassette comprising a DNAsequence of interest which encodes a structural protein of HIV.

In certain embodiments, the DNA sequence of interest encodes a proteinassociated with cancer.

In certain embodiments, the DNA sequence of interest encodes a proteinassociated with immune maturation, regulation of immune responses, orregulation of autoimmune responses. In a specific embodiment, theprotein is APECED.

In a specific embodiment, the DNA sequence of interest is the Aire gene.

In certain embodiments, the DNA sequence of interest encodes a proteinthat is defective in any hereditary single gene disease.

In certain embodiments, the DNA sequence of interest encodes amacromolecular drug.

In certain embodiments, the DNA sequence of interest encodes a cytokine.In certain specific embodiments, the cytokine is an interleukin selectedfrom the group consisting of IL1, IL2, IL4, IL6 and IL12. In certainother specific embodiments, the DNA sequence of interest encodes aninterferon.

In certain embodiments, the DNA sequence of interest encodes abiologically active RNA molecule. In certain specific embodiments, thebiologically active RNA molecule is selected from the group consistingof inhibitory antisense and ribozyme molecules. In certain specificembodiments, the inhibitory antisense or ribozyme molecules antagonizethe function of an oncogene.

A vector of the invention is suitable for the use for the production ofa therapeutic macromolecular agent in vivo.

In certain embodiments, the invention provides a vector for use as amedicament.

In certain embodiments, the invention provides a vector for use as acarrier vector for a gene, genes, or a DNA sequence or DNA sequences ofinterest, such as a gene, genes, or a DNA sequence or DNA sequencesencoding a protein or peptide of an infectious agent, a therapeuticagent, a macromolecular drug, or any combination thereof.

In certain specific embodiments, the invention provides a vector for useas a medicament for treating inherited or acquired genetic defects.

In certain embodiments, the invention provides a vector for use as atherapeutic DNA vaccine against an infectious agent.

In certain embodiments, the invention provides a vector for use as atherapeutic agent.

The invention further relates to methods for providing a protein to asubject, said method comprising administering to the subject a vector ofthe invention, wherein said vector (i) further comprises a second DNAsequence encoding the protein to be provided to the subject, whichsecond DNA sequence is operably linked to a second promoter, and (ii)does not encode Bovine Papilloma Virus protein E1, and wherein saidsubject does not express Bovine Papilloma Virus protein E1.

The invention further relates to methods for inducing an immune responseto a protein in a subject, said method comprising administering to thesubject a vector of the invention wherein said vector (i) furthercomprises a second DNA sequence encoding said protein, which second DNAsequence is operably linked to a second promoter, and (ii) does notencode Bovine Papilloma Virus protein E1, and wherein said subject doesnot express Bovine Papilloma Virus protein E1.

The invention further relates to methods for treating an infectiousdisease in a subject in need of said treatment, said method comprisingadministering to said subject a therapeutically effective amount of avector of the invention, wherein the DNA sequence of interest encodes aprotein comprising an immunogenic epitope of an infectious agent.

The invention further relates to methods for treating an inherited oracquired genetic defect in a subject in need of said treatment, saidmethod comprising: administering to said subject a therapeuticallyeffective amount of a vector of the invention, wherein said DNA sequenceof interest encodes a protein which is affected by said inherited oracquired genetic defect.

The invention further relates to methods for expressing a DNA sequencein a subject, said method comprising administering a vector of theinvention to said subject.

The invention further relates to methods for expressing a DNA sequencein a subject, treating an inherited or acquired genetic defect, treatingan infectious disease, inducing an immune-response to a protein, andproviding a protein to a subject, wherein the vector of the inventiondoes not encode Bovine Papilloma Virus protein E1, and wherein saidsubject does not express Bovine Papilloma Virus protein E1.

In certain embodiments, a vector of the invention is used for productionof a protein encoded by said DNA sequence of interest in a cell or anorganism.

The invention further provides a method for the preparation of a vectorof claim 1, 2, or 17 comprising: (a) cultivating a host cell containingsaid vector and (b) recovering the vector. In a specific embodiment, themethod for preparing a vector of the invention further comprises beforestep (a) a step of transforming said host cell with said vector. Incertain specific embodiments, the host cell is a prokaryotic cell. In aspecific embodiment, the host cell is an Escherichia coli.

The invention further relates to a host cell that is characterized bycontaining a vector of the invention. In certain embodiments, the hostcell is a bacterial cell. In a certain other embodiments, the host cellis a mammalian cell.

The invention further relates to carrier vectors containing a vector ofthe invention.

The invention further relates to a pharmaceutical composition comprisinga vector of the invention and a suitable pharmaceutical vehicle.

The invention further relates to a DNA vaccine containing a vector ofthe invention.

The invention further relates to a gene therapeutic agent containing avector of the invention.

The invention further relates to a method for the preparation of a DNAvaccine, said method comprising combining a vector of the invention witha suitable pharmaceutical vehicle.

The invention further relates to a method for the preparation of anagent for use in gene therapy, said method comprising combining a vectorof the invention with a suitable pharmaceutical vehicle.

4. DESCRIPTION OF THE FIGURES

FIG. 1 shows the schematic map of plasmid super6.

FIG. 2 shows the schematic map of plasmid VI.

FIG. 3 shows the schematic map of plasmid II.

FIG. 4 shows the expression of the Nef and E2 proteins from the vectorssuper6, super6wt, VI, VIwt, and II in Jurkat cells.

FIG. 5 shows the schematic map of plasmid product1.

FIG. 6A shows the schematic map of the plasmids NNV-1 and NNV-2 and FIG.6B shows the schematic map of plasmid and NNV-2wt.

FIG. 7 shows the expression of the Nef protein from the plasmids NNV-1,NNV-2, NNV-1wt, NNV-2-wt, super6, and super6wt in Jurkat cells.

FIG. 8 shows the expression of the Nef and E2 proteins from the plasmidsNNV-2-wt, NNV-2-wtFS, and product I in Jurkat cells.

FIG. 9 shows the expression of the Nef and E2 proteins from the plasmidsNNV-2-wt, NNV-2-wtFS, and product I in P815 cells.

FIG. 10 shows the expression of the Nef and E2 proteins from theplasmids NNV-2-wt, NNV-2-wtFS, and product I in CHO cells.

FIG. 11 shows the expression of the Nef protein from the plasmidsNNV-2-wt, NNV-2-wtFS, and product I in RD cells.

FIG. 12 shows the expression of the RNA molecules NNV-2wt in CHO, Jurkatcells, and P815 cells.

FIG. 13 shows the stability of NNV-2wt in bacterial cells.

FIG. 14 shows the Southern blot analysis of stability of the NNV-2wt asnon-replicating episomal element in CHO and Jurkat cell lines.

FIG. 15 shows that the vectors NNV2wt, NNV2wtFS and product1 are unableto HPV-11 replication factor-dependent replication.

FIG. 16 shows the schematic map of the plasmid 2wtd1EGFP.

FIG. 17 shows the schematic map of the plasmid gf10bse2

FIG. 18 shows the schematic map of the plasmid 2wtd1EGFPFS.

FIG. 19 shows the schematic map of the plasmid NNVd1EGFP.

FIG. 20 shows the growth curves of the Jurkat cells transfected with theplasmids 2wtd1EGFP, 2wtd1EGFPFS, NNVd1EGFP or with carrier DNA only.

FIG. 21 shows the growth curves of the Jurkat cells transfected with theplasmids 2wtd1EGFP, 2wtd1EGFPFS, gf10bse2 or with carrier DNA only.

FIG. 22 shows the change in the percentage of d1EGFP positive cells in apopulation of Jurkat cells transfected with the vectors 2wtd1EGFP,2wtd1EGFPFS or NNVd1EGFP.

FIG. 23 shows the change in percentage of the d1EGFP positive cells in apopulation of Jurkat cells transfected with the vectors 2wtd1EGFP,2wtd1EGFPFS or gf10bse2.

FIG. 24 shows the change in the number of d1EGFP expressing cells in apopulation of Jurkat cells transfected with the vectors 2wtd1EGFP,2wtd1EGFPFS or NNVd1EGFP.

FIG. 25 shows the change in the number of d1EGFP expressing cells in apopulation of Jurkat cells transfected with the vectors 2wtd1EGFP,2wtd1EGFPFS or gf10bse2.

FIG. 26. T-cell responses towards recombinant Nef proteins (5micrograms/well), measured by T-cell proliferation in five patientsimmunized with 1 microgram of GTU-Nef.

FIG. 27. T-cell responses towards recombinant Nef proteins (5micrograms/well), measured by T-cell proliferation in five patientsimmunized with 20 micrograms of GTU-Nef.

FIG. 28. T-cell responses towards recombinant Nef proteins (5micrograms/well), measured by T-cell proliferation in patient# 1immunized with 1 microgram of GTU-Nef. The results are given asstimulation index of the T-cell proliferation assay (Nef SI) and asIFN-Gamma secretion to the supernatant.

FIG. 29. (A) plasmid pEBO LPP; (B) plasmid s6E2d1EGFP; (C) plasmidFRE2d1EGFP

FIG. 30. Plasmid FREBNAd1EGFP

FIG. 31. Vectors did not interfere with cell proliferation

FIG. 32. Vectors were maintained in the cells with different kinetics

FIG. 33. Change of the number of d1EGFP expressing cells in time intransfected total population of cells

FIG. 34. Change of the number of d1EGFP expressing cells in time intransfected total population of cells. (A) human embryonic cell line293; (B) mouse cell line 3T6

FIG. 35. Nef and E2 antibody response

FIG. 36. Rev and Tat antibody response

FIG. 37. Gag and CTL response

FIG. 38. (A) GTU-1; (B) GTU-2Nef; (C) GTU-3Nef; (D) super6 wtd1EGFP; (E)FREBNAd1EGFP; (F) E2BSEBNAd1EGFP; (G) NNV-Rev

FIG. 39. (A) pNRT; (B) pTRN; (C) pRTN; (D) pTNR; (E) pRNT; (F) p2TRN;(G) p2RNT; (H) p3RNT; (I) pTRN-iE2-GMCSF; (J) pTRN-iMG-GMCSF

FIG. 40. (A) pMV1NTR; (B) pMV2NTR; (C) pMV1N11TR; (D) pMV2N11TR

FIG. 41. (A) pCTL; (B) pdgag; (C) psynp17/24; (D) poptp17/24; (E)p2mCTL; (F) p2optp17/24; (G) p3mCTL; (H) p3optp17/24

FIG. 42. (A) pTRN-CTL; (B) pRNT-CTL; (C) pTRN-dgag; (D) pTRN-CTL-dgag;(E) pRNT-CTL-dgag; (F) pTRN-dgag-CTL; (G) pRNT-dgag-CTL; (H)pTRN-optp17/24-CTL; (I) pTRN-CTL-optp17/24; (J) pRNT-CTL-optp17/24; (K)p2TRN-optp17/24-CTL; (L) p2RNT-optp17/24-CTL; (M) p2TRN-CTL-optp17/24;(N) p2RNT-CTL-optp17/24; (O) p2TRN-CTL-optp17/24-iE2-mGMCSF; (P)p2RNT-CTL-optp17/24-iE2-mGMCSF; (O) p3TRN-CTL-optp17/24; (R)p3RNT-CTL-optp17/24; (S) p3TRN-CTL-optp17/24-iE2-mGMCSF; (T)p3RNT-CTL-optp17/24-iE2-mGMCSF; (U) FREBNA-RNT-CTL-optp17/24; (V)super6wt-RNT-CTL-optp17/24; (W) E2BSEBNA-RNT-CTL-optp17/24; (X)pCMV-RNT-CTL-optp17/24

FIG. 43. Analysis of expression of the multireg antigens.

FIG. 44. Analysis of expression of the multireg antigens comprised ofimmunodominant parts of the proteins.

FIG. 45. Analysis of intracellular localization of multireg antigens byimmunofluorescence.

FIG. 46. Analysis of expression of the gag coded structural proteins andthe CTL multi-epitope.

47. The p17/24 protein localization in membranes of RD cells.

FIG. 48. Analysis expression of the dgag and CTL containing multigenesin Cos-7 cells.

FIG. 49. Western blot analyses of multiHIV antigens expressed in Jurkatcells.

FIG. 50. Analysis of the expression of the TRN-CTL-optp17/24 andRNT-CTL-optp17/24 antigens as well E2 protein from the GTU-1, GTU-2 andGTU-3 vector.

FIG. 51. The maintenance of the multiHIV antigen expression fromdifferent vectors.

FIG. 52. Intracellular localization of the multiHIV antigens in RDcells.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 Vectors of the Invention

The present invention is based on the unexpected finding that expressionvectors, which carry (A) an expression cassette of a gene of anuclear-anchoring protein that binds both to (i) a specific DNA sequenceand (ii) to a suitable nuclear component and (B) a multimerized DNAbinding sequence for said nuclear-anchoring protein are capable ofspreading in a proliferating cell population. Such nuclear-anchoringproteins include, but are not limited to, chromatin-anchoring proteins,such as the Bovine Papilloma Virus type 1 E2 protein (BPV1 E2; SEQ IDNO: 50). The DNA binding sequences can be, but are not limited to,multimerized E2 binding sites. On the basis of prior art, it could notbe expected that a segregation/partitioning function of, for instance,the papilloma viruses could be expressed separately and that an additionof such segregation/partitioning function to the vaccine vectors wouldassure the distribution of the vector in the proliferating cellpopulation. Additionally, on the basis of the prior art, it could nothave been expected that functional vectors acting independently of thereplication origin can be constructed.

The term “nuclear-anchoring protein” as used in the present inventionrefers to a protein, which binds to a specific DNA sequence and capableof providing a nuclear compartmentalization function to the vector,i.e., to a protein, which is capable of anchoring or attaching thevector to a specific nuclear compartment. In certain embodiments of theinvention, the nuclear-anchoring protein is a natural protein. Examplesof such nuclear compartments are the mitotic chromatin or mitoticchromosomes, the nuclear matrix, nuclear domains like ND10 and POD etc.Examples of nuclear-anchoring proteins are the Bovine Papilloma Virustype 1 (BPV1) E2 protein, EBNA1 (Epstein-Barr Virus Nuclear Antigen 1;SEQ ID NO: 52), and High Mobility Group (HMG) proteins etc. The term“functional equivalent of a nuclear-anchoring protein” as used in thepresent invention refers to a protein or a polypeptide of natural ornon-natural origin having the properties of the nuclear-anchoringprotein.

In certain other embodiments of the invention, the nuclear-anchoringprotein of the invention is a recombinant protein. In certain specificembodiments of the invention, the nuclear-anchoring protein is a fusionprotein, a chimeric protein, or a protein obtained by molecularmodeling. A fusion protein, or a protein obtained by molecular modelingin connection with the present invention is characterized by its abilityto bind to a nuclear component and by its ability to bindsequence-specifically to DNA. In a preferred embodiment of theinvention, such a fusion protein is encoded by a vector of the inventionwhich also contains the specific DNA sequence to which thefusion/chimeric protein binds. Nuclear components include, but are notlimited to chromatin, the nuclear matrix, the ND10 domain and POD. Inorder to reduce the risk of interference with the expression of genesendogenous to the host cell, the DNA binding domain and thecorresponding DNA sequence is preferably non-endogenous to the hostcell/host organism. Such domains include, but are not limited to, theDNA binding domain of the Bovine Papilloma Virus type 1 (BPV1) E2protein (SEQ ID NO: 50), Epstein-Barr Virus Nuclear Antigen 1 (EBNA1;SEQ ID NO: 52), and High Mobility Group (HMG) proteins (HMG box).

The vector of the invention can further comprise a “DNA sequence ofinterest”, that encodes a protein (including a peptide or polypeptide),e.g., that is an immunogen or a therapeutic. In certain embodiments ofthe invention, the DNA sequence of interest encodes a biologicallyactive RNA molecule, such as an antisense RNA molecule or a ribozyme.

The expression vectors of the invention carrying an expression cassettefor a gene of a nuclear-anchoring protein and multimerized binding sitesfor said nuclear-anchoring protein spread in a proliferating host cellpopulation. This means that a high copy-number of vectors or plasmidsare delivered into the target cells and the use of thesegregation/partitioning function of the nuclear-anchoring protein andits multimerized binding sites assures the distribution of the vector tothe daughter cells during cell division.

The vector of the invention lacks a papilloma virus origin ofreplication. Further, in a preferred embodiment, the vector of theinvention lacks an origin of replication functional in a mammalian cell.The omission of a papilloma virus origin of replication or a mammalianorigin of replication constitutes an improvement over prior art vectorsfor several reasons. (1) Omission of the origin of replication reducesthe size of the vector of the invention compared to prior art vectors.Such a reduction in size increases the stability of the vector andfacilitates uptake by the host cell. (2) Omission of the origin ofreplication reduces the risk for recombination with the host cell'sgenome, thereby reducing the risk of unwanted side effects. (3) Theomission of the origin of replication allows to control the dosagesimply by adjusting the amount of vector administered. In contrast, witha functioning origin of replication, replication of the vector has to betaken into consideration when determining the required dosage. (4) Ifthe vector is not administered to a host organism continually, the lackof an origin of replication allows the host organism to clear itself ofthe vector, thus providing more control over the levels of DNA sequencesto be expressed in the host organism. Further, the ability of theorganism to clear itself of the vector will be advantageous if thepresence of the vector is required only during the course of a therapybut is undesirable in a healthy individual.

The gene of a nuclear-anchoring protein useful in the vectors of thepresent invention can be any suitable DNA sequence encoding a natural orartificial protein, such as a recombinant protein, a fusion protein or aprotein obtained by molecular modeling techniques, having the requiredproperties. Thus the gene of a natural nuclear-anchoring protein, whichcontains a DNA binding domain capable of binding to a specific DNAsequence and a functional domain capable of binding to a nuclearcomponent, can be that of a viral protein, such as the E2 protein ofBovine Papilloma Virus or the EBNA1 (Epstein-Barr Virus NuclearAntigen 1) of the Epstein-Barr Virus, a eukaryotic protein such a one ofthe High Mobility Group (HMG) proteins or a like protein, or aprokaryotic protein. Alternatively, the gene of a nuclear-anchoringprotein, which contains a DNA binding domain capable of binding to aspecific DNA sequence and a functional domain capable of binding to anuclear component, can also be comprised of DNA sequences, which encodea domain from a cellular protein having the ability to attach to asuitable nuclear structure, such as to mitotic chromosomes, the nuclearmatrix or nuclear domains like ND10 or POD.

Alternatively, the DNA sequence, which encodes a non-natural orartificial protein, such as a recombinant protein or a fusion protein ora protein obtained by molecular modeling, which contains a DNA bindingdomain capable of binding to a specific DNA sequence of, e.g., apapilloma virus, such as the DNA binding domain of the E2 protein of theBPV1, but in which the N-terminus of the nuclear-anchoring protein, e.g.that of the E2 protein, has been replaced with domains of any suitableprotein of similar capacity, for example, with the N-terminal domain ofEpstein-Barr Virus Nuclear Antigen 1 sequence, can be used. Similarly,DNA sequences, which encode a recombinant protein or a fusion protein,which contains a functional domain capable of binding to a nuclearcomponent, e.g., the N-terminal functional domain of a papilloma virus,such as the E2 protein of the BPV1, but in which the C-terminalDNA-binding dimerization domain of the nuclear-anchoring protein, e.g.,that of the E2 protein, has been replaced with domains of any protein ofa sufficient DNA-binding strength, e.g., the DNA binding domain of theBPV-1 E2 protein and the EBNA-1, can be used.

In a preferred embodiment of the invention, the nuclear-anchoringprotein is a chromatin-anchoring protein, which contains a DNA bindingdomain, which binds to a specific DNA sequence, and a functional domaincapable of binding to mitotic chromatin. A preferred example of such achromatin-anchoring protein and its multimerized binding sites useful inthe present invention are the E2 protein of Bovine Papilloma Virus type1 and E2 protein multimerized binding sites. In the case of E2, themechanism of the spreading function is due to the dual function of theE2 protein: the capacity of the E2 protein to attach to mitoticchromosomes through the N-terminal domain of the protein and thesequence-specific binding capacity of the C-terminal domain of the E2protein, which assures the tethering of vectors, which contain amultimerized E2 binding site, to mitotic chromosomes. Asegregation/partitioning function is thus provided to the vectors.

In another preferred embodiment of the invention, the expressioncassette of a gene of the chromatin-anchoring protein comprises a geneof any suitable protein of cellular, viral or recombinant origin havinganalogous properties to E2 of the BPV1, i.e., the ability to attach tothe mitotic chromatin through one domain and to cooperatively bind DNAthrough another domain to multimerized binding sites specific for thisDNA binding domain.

In a specific embodiment, sequences obtained from BPV1, are used in thevectors of the present invention, they are extensively shortened in sizeto include just two elements from BPV1. First, they include the E2protein coding sequence transcribed from a heterologous eukaryoticpromoter and polyadenylated at the heterologous polyadenylation site.Second, they include E2 protein multiple binding sites incorporated intothe vector as a cluster, where the sites can be as head-to-tailstructures or can be included into the vector by spaced positioning.Both of these elements are necessary and, surprisingly, sufficient forthe function of the vectors to spread in proliferating cells. Similarly,when DNA sequences based of other suitable sources are used in thevectors of the present invention, the same principles are applied.

According to the present invention, the expression cassette of a gene ofa nuclear-anchoring protein, which contains a DNA binding domain capableof binding to a specific DNA sequence and a functional domain capable ofbinding to a nuclear component, such as an expression cassette of a geneof a chromatin-anchoring protein, like BPV1 E2, comprises a heterologouseukaryotic promoter, the nuclear-anchoring protein coding sequence, suchas a chromatin-anchoring protein coding sequence, for instance the BPV1E2 protein coding sequence, and a poly A site. Different heterologous,eukaryotic promoters, which control the expression of thenuclear-anchoring protein, can be used. Nucleotide sequences of suchheterologous, eukaryotic promoters are well known in the art and arereadily available. Such heterologous eukaryotic promoters are ofdifferent strength and tissue-specificity. In a preferred embodiment,the nuclear anchoring protein is expressed at low levels.

The multimerized DNA binding sequences, i.e., DNA sequences containingmultimeric binding sites, as defined in the context of the presentinvention, are the region, to which the DNA binding dimerization domainbinds. The multimerized DNA binding sequences of the vectors of thepresent invention can contain any suitable DNA binding site, providedthat it fulfills the above requirements.

In a preferred embodiment, the multimerized DNA binding sequence of avector of the present invention can contain any one of known 17different affinity E2 binding sites as a hexamer or a higher oligomer,as a octamer or a higher oligomer, as a decamer or higher oligomer.Oligomers containing different E2 binding sites are also applicable.Specifically preferred E2 binding sites useful in the vectors of thepresent invention are the BPV1 high affinity sites 9 and 10, affinitysite 9 being most preferred. When a higher oligomer is concerned, itssize is limited only by the construction circumstances and it maycontain from 6 to 30 identical binding sites. Preferred vectors of theinvention contain 10 BPV-1 E2 binding sites 9 in tandem. When themultimerized DNA binding sequences are comprised of different E2 bindingsites, their size and composition is limited only by the method ofconstruction practice. Thus they may contain two or more different E2binding sites attached to a series of 6 to 30, most preferably 10, E2binding sites.

The Bovine Papilloma Virus type 1 genome (SEQ ID NO: 49) contains 17 E2protein binding sites which differ in their affinity to E2. The E2binding sites are described in Li et al. [Genes Dev 3 (4) (1989)510-526], which is incorporated by reference in its entirety herein.

Alternatively, the multimerized DNA binding sequences may be composed ofany suitable multimeric specific sequences capable of inducing thecooperative binding of the protein to the plasmid, such as those of theEBNA1 or a suitable HMG protein. 21×30 bp repeats of binding sites forEBNA-1 are localized in the region spanning from nucleotide position7421 to nucleotide position 8042 of the Epstein-Barr virus genome (SEQID NO:51). These EBNA-1 binding sites are described in the followingreferences: Rawlins et al., Cell 42 (3) (1985) 859-868; Reisman et al.,Mol Cell Biol 5 (8) (1985) 1822-1832; and Lupton and Levine, Mol CellBiol 5 (10) (1985) 2533-2542, all three of which are incorporated byreference in their entireties herein.

The position of the multimerized DNA binding sequences relative to theexpression cassette for the DNA binding dimerization domain is notcritical and can be any position in the plasmid. Thus the multimerizedDNA binding sequences can be positioned either downstream or upstreamrelative to the expression cassette for the gene of interest, a positionclose to the promoter of the gene of interest being preferred.

The vectors of the invention also contain, where appropriate, a suitablepromoter for the transcription of the gene or genes or the DNA sequencesof interest, additional regulatory sequences, polyadenylation sequencesand introns. Preferably the vectors may also include a bacterial plasmidorigin of replication and one or more genes for selectable markers tofacilitate the preparation of the vector in a bacterial host and asuitable promoter for the expression the gene for antibiotic selection.

The selectable marker can be any suitable marker allowable in DNAvaccines, such a kanamycin or neomycin, and others. In addition, otherpositive and negative selection markers can be included in the vectorsof the invention, where applicable.

The vectors of the present invention only comprise the DNA sequences,for instance BPV1 DNA sequences, which are necessary and sufficient forlong-term maintenance. All superfluous sequences, which may induceadverse reactions, such as oncogenic sequences, have been deleted. Thusin preferred vectors of the invention the E2 coding sequence is modifiedby mutational analysis so that this expresses only E2 protein andoverlapping E3, E4 and E5 sequences have been inactivated by theintroduction of mutations, which inactivate the translation from OpenReading Frames for E3, E4 and E5. The vector of the invention does notcontain a papilloma virus origin of replication. Preferably, the vectorof the invention further does not contain an origin of replicationfunctional in a mammalian cell or a mammal.

Furthermore, the vectors of the present invention are not host specific,since the expression of the nuclear-anchoring protein, such as the E2protein, is controlled by non-native or heterologous promoters.Depending on the particular promoter chosen, these promoters may befunctional in a broad range of mammalian cells or they can be cell ortissue specific. Examples of promoters for the nuclear-anchoringprotein, such as for the E2 protein, useful in the vectors of thepresent invention are thymidine kinase promoters, Human CytomegalovirusImmediate Early Promoter, Rous Sarcoma Virus LTR and like. For theexpression of the gene of interest, preferred promoters are strongpromoters assuring high levels of expression of the gene of interest, anexample for such a promoter is the Human Cytomegalovirus Immediate EarlyPromoter.

5.2 The Vectors of the Invention as Vehicles for Expression of a DNASequence of Interest

A gene, genes or a DNA sequence or DNA sequences to be expressed via avector of the invention can be any DNA sequence of interest, whoseexpression is desired. Thus the vectors may contain a gene or genes or aDNA sequence or DNA sequences from infectious microbial pathogens, suchas viruses, against which live attenuated vaccines or inactivatedvaccines cannot be prepared or used. Such DNA sequences of interestinclude genes or DNA sequences from viruses, such as HumanImmunodeficiency Virus (HIV), Herpex Simplex Virus (HSV), Hepatitis CVirus, Influenzae Virus, Enteroviruses etc.; intracellular bacterial,such as Chlamydia trachomatis, Mycobacterium tuberculosis, Mycoplasmapneumonia etc.; extracellular bacteria, such as Salmonella; or fungi,such as Candida albigans.

In a preferred embodiment of the invention, the vectors contain a geneencoding early regulatory proteins of HIV, i.e. the nonstructuralregulatory proteins Nef, Tat or Rev, preferably Nef. In anotherpreferred embodiment of the invention the vectors of the inventioncontain genes encoding structural proteins of the HIV. In anotherpreferred embodiment the vectors of the present invention contain two ormore genes encoding any combination of early regulatory proteins and/orstructural proteins of HIV. Illustrative examples of such combinationsare a combination of a gene encoding the Nef protein and a DNA sequenceencoding the Tat protein, possibly together with a DNA sequence encodingouter envelope glycoprotein of HIV, gp120/gp160 or a combination of anyimmunogenic epitopes of the proteins of pathogens incorporated intoartificial recombinant protein.

Alternatively, the vectors of the invention may contain genes or DNAsequences for inherited or acquired genetic defects, such as sequencesof differentiation antigens for melanoma, like a Tyrosinase A codingsequence or a coding sequence of beta-catenins.

In a preferred embodiment of the invention, the vectors contain a geneencoding proteins relating to cancer or other mutational diseases,preferably diseases related to immune maturation and regulation ofimmune response towards self and nonself, such as the APECED gene.

In another preferred embodiment of the invention, the vectors containany DNA sequence coding for a protein that is defective in anyhereditary single gene hereditary disease.

In another preferred embodiment of the invention, the vectors containany DNA sequence coding for a macromolecular drug to be delivered andproduced in vivo.

The method of the invention for the preparation of the vectors of theinvention comprises the following steps: (A) cultivating a host cellcontaining a vector of the invention, and (B) recovering the vector. Incertain specific embodiments, step (A) is preceded by transforming ahost cell with a vector of the invention.

The vectors of the invention are preferably amplified in a suitablebacterial host cell, such as Escherichia coli. The vectors of theinvention are stable and replicate at high copy numbers in bacterialcells. If a vector of the invention is to be amplified in a bacterialhast cell, the vector of the invention contains a bacterial origin ofreplication. Nucleotide sequences of bacterial origins of replicationare well known to the skilled artisan and can readily be obtained.

Upon transfection into a mammalian host in high copy number, the vectorspreads along with cell divisions and the number of cells carrying thevector increases without the replication of the vector, each cell beingcapable of expressing the protein of interest.

The vectors of the invention result in high expression of the desiredprotein. For instance, as demonstrated in Examples 4, 7-10: a highexpression of the Nef protein of the HIV, green fluorescent protein(EGFP) and the AIRE protein could be demonstrated in many different celllines and the data indicate that not only the number of positive cells,but the quantity of the protein encoded by the gene of interest isincreasing in time.

The vectors of the invention also induce both humoral and cellularresponse as demonstrated in Examples 9 and 10. The results indicate thatthe vectors of the present invention can effectively be used as DNAvaccines.

The vaccines of the present invention contain a vector of the presentinvention or a mixture of said vectors in a suitable pharmaceuticalcarrier. The vaccine may for instance contain a mixture of vectorscontaining genes for the three different regulatory proteins of the HIVand/or structural proteins of the HIV.

The vaccines of the invention are formulated using standard methods ofvaccine formulation to produce vaccines to be administered by anyconventional route of administration, i.e. intramuscularly,intradermally and like.

The vectors of the invention may contain the ISS stimulatory sequencesin order to activate the immune response of the body.

The vaccines of the invention can be used in a conventional preventivemanner to protect an individual from infections, Alternatively, thevaccines of the invention can be used as therapeutical vaccines,especially in the case of viral infections, together with a conventionalmedication.

As mentioned above, the vectors of the present invention carrying themechanism of spreading in the host cell find numerous applications asvaccines, in gene therapy, in gene transfer and as therapeuticimmunogens. The vectors of the invention can be used to deliver a normalgene to a host having a gene defect, thus leading to a cure or therapyof a genetic disease. Furthermore, the vectors can deliver genes ofimmunogenic proteins of foreign origin, such as those from microbes orautologous tumor antigens, to be used in the development of vaccinesagainst microbes or cancer. Furthermore, the vectors of the inventioncan deliver suitable genes of marker substances to nucleus, to be usedin studies of cellular function or in diagnostics. Finally, the vectorsof the invention can be used to specifically deliver a gene ofmacromolecular drug to the nucleus, thus enabling the development ofnovel therapeutic principles to treat and cure diseases, where theexpression of the drug in the site of action, the cell nucleus, is ofimportance. These drugs can be chemical macromolecules, such as anyproteins or polypeptides with therapeutic or curative effect, whichinterfere with any of the nuclear mechanisms, such as the replication ortranscription or the trans-port of substances to and from the nucleus.

Specifically, the vectors of the present invention can be used for theexpression of the specific cytokines, like interleukines (IL1, IL2, IL4,IL6, IL12 and others) or interferon, with the aim of modulating thespecific immune responses of the organism (immunotherapy) againstforeign antigens or boosting of the activity of the immune systemagainst the mutated self-antigens. The vectors of the present inventionare also useful in complementing malfunctioning of the brain due to theloss of specific dopamine-ergic neurons leading to the irreversibleneurodegeneration, which is cause for Parkinson's disease, by expressinggenes involved into synthesis of dopamine, like tyrosine hydroxylase, aswell as other genes deficiency of which would have the similar effect.The vectors of the present invention are also useful for the expressionof proteins and peptides regulating the brain activity, like dopaminereceptors, CCK-A and CCK-B receptors, as well as neurotrophic factors,like GDNF, BDNF and other proteins regulating the brain activity.Further, the vectors of the present invention are useful for a long-termexpression of factor IX in hepatocytes and alfa1-antitrypsin in musclecells with the aim of complementing respective deficiencies of theorganism.

5.3 Target Diseases and Disorders

In certain embodiments, a vector of the invention is used as a vaccine.In certain embodiments, a vector of the invention contains a DNAsequence of interest that encodes a protein or a peptide. Uponadministering of such a vector to a subject, the protein or peptideencoded by the DNA sequence of interest is expressed and stimulates animmune response specific to the protein or peptide encoded by the DNAsequence of interest.

In specific embodiments, the vector of the invention is used to treatand/or prevent an infectious disease and/or a condition caused by aninfectious agent. Such diseases and conditions include, but are notlimited to, infectious diseases caused by bacteria, viruses, fungi,protozoa, helminths, and the like. In a more specific embodiment of theinvention, the infectious disease is Acquired Immunodeficiency Syndrome.

Preferably, where it is desired to treat or prevent viral diseases, DNAsequences encoding molecules comprising epitopes of known viruses areused. For example, such DNA sequences encoding antigenic epitopes may beprepared from viruses including, but not limited to, hepatitis type A,hepatitis type B, hepatitis type C, influenza, varicella, adenovirus,herpes simplex type I (HSV-I), herpes simplex type II (HSV-II),rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytialvirus, papilloma virus, papova virus, cytomegalovirus, echinovirus,arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus,rubella virus, polio virus, human immunodeficiency virus type I (HIV-I),and human immunodeficiency virus type II (HIV-II).

Preferably, where it is desired to treat or prevent bacterialinfections, DNA sequences encoding molecules comprising epitopes ofknown bacteria are used. For example, such DNA sequences encodingantigenic epitopes may be prepared from bacteria including, but notlimited to, mycobacteria rickettsia, mycoplasma, neisseria andlegionella.

Preferably, where it is desired to treat or prevent protozoalinfections, DNA sequences encoding molecules comprising epitopes ofknown protozoa are used. For example, such DNA sequences encodingantigenic epitopes may be prepared from protozoa including, but notlimited to, leishmania, kokzidioa, and trypanosoma.

Preferably, where it is desired to treat or prevent parasiticinfections, DNA sequences encoding molecules comprising epitopes ofknown parasites are used. For example, such DNA sequences encodingantigenic epitopes may be prepared from parasites including, but notlimited to, chlamydia and rickettsia.

In other specific embodiments, the vector of the invention is used totreat and/or prevent a neoplastic disease in a subject. In theseembodiments, the DNA sequence of interest encodes a protein or peptidethat is specific to or associated with the neoplastic disease. By way ofnon-limiting example, the neoplastic disease can be a fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenström's macroglobulinemia, and heavychain disease, etc.

In certain other embodiments of the invention, the DNA sequence ofinterest encodes a protein that is non-functional or malfunctioning dueto an inherited disorder or an acquired mutation in the gene encodingthe protein. Such genetic diseases include, but are not limited to,metabolic diseases, e.g., Atherosclerosis (affected gene: APOE); cancer,e.g., Familial Adenomatous Polyposis Coli (affected gene: APC gene);auto-immune diseases, e.g., autoimmunepolyendocrinopathy-candidosis-ectodermal dysplasia (affected gene:APECED); disorders of the muscle, e.g., Duchenne musculardystrophyvaccines (affected gene: DMD); diseases of the nervous system,e.g., Alzheimer's Disease (affected genes: PS1 and PS2).

In even other embodiments, the vectors of the invention are used totreat and/or prevent diseases and disorders caused by pathologicallyhigh activity of a protein. In these embodiments of the invention, theDNA sequence of interest encodes an antagonist of the overactiveprotein. Such antagonists include, but are not limited to, antisense RNAmolecules, ribozymes, antibodies, and dominant negative proteins. Inspecific embodiments of the invention, the DNA sequence of interestencodes an inhibitor of an oncogene.

In certain embodiments, the DNA sequence of interest encodes a moleculethat antagonizes neoplastic growth. In specific embodiments of theinvention, the DNA sequence of interest encodes a tumor suppressor, suchas, but not limited to, p53. In other specific embodiments, the DNAsequence of interest encodes an activator of apoptosis, such as but notlimited to, a Caspase.

The invention provides methods, whereby a DNA sequence of interest isexpressed in a subject. In certain embodiments, a vector containing oneor more expression cassettes of a DNA sequence of interest isadministered to the subject, wherein the subject does not express theBovine Papilloma Virus E1 protein.

5.4 Therapeutic Methods for Use with the Invention

5.4.1 Recombinant DNA

In various embodiments of the invention, the vector of the inventioncomprises one or more expression cassettes comprising a DNA sequence ofinterest. The DNA sequence of interest can encode a protein and/or abiologically active RNA molecule. In either case, the DNA sequence isinserted into the vector of the invention for expression in recombinantcells or in cells of the host in the case of gene therapy.

An expression cassette, as used herein, refers to a DNA sequence ofinterest operably linked to one or more regulatory regions orenhancer/promoter sequences which enables expression of the protein ofthe invention in an appropriate host cell. “Operably-linked” refers toan association in which the regulatory regions and the DNA sequence tobe expressed are joined and positioned in such a way as to permittranscription, and in the case of a protein, translation.

The regulatory regions necessary for transcription of the DNA sequenceof interest can be provided by the vector of the invention. In acompatible host-construct system, cellular transcriptional factors, suchas RNA polymerase, will bind to the regulatory regions of the vector toeffect transcription of the DNA sequence of interest in the hostorganism. The precise nature of the regulatory regions needed for geneexpression may vary from host cell to host cell. Generally, a promoteris required which is capable of binding RNA polymerase and promoting thetranscription of an operably-associated DNA sequence. Such regulatoryregions may include those 5′-non-coding sequences involved withinitiation of transcription and translation, such as the TATA box,capping sequence, CAAT sequence, and the like. The non-coding region 3′to the coding sequence may contain transcriptional terminationregulatory sequences, such as terminators and polyadenylation sites.

Both constitutive and inducible regulatory regions may be used forexpression of the DNA sequence of interest. It may be desirable to useinducible promoters when the conditions optimal for growth of the hostcells and the conditions for high level expression of the DNA sequenceof interest are different. Examples of useful regulatory regions areprovided below (section 5.4.4).

In order to attach DNA sequences with regulatory functions, such aspromoters, to the DNA sequence of interest or to insert the DNA sequenceof interest into the cloning site of a vector, linkers or adaptersproviding the appropriate compatible restriction sites may be ligated tothe ends of the cDNAs by techniques well known in the art [Wu et al.,Methods in Enzymol 152 (1987) 343-349). Cleavage with a restrictionenzyme can be followed by modification to create blunt ends by digestingback or filling in single-stranded DNA termini before ligation.Alternatively, a desired restriction enzyme site can be introduced intoa fragment of DNA by amplification of the DNA by use of PCR with primerscontaining the desired restriction enzyme site.

The vector comprising a DNA sequence of interest operably linked to aregulatory region (enhancer/promoter sequences) can be directlyintroduced into appropriate host cells for expression of the DNAsequence of interest without further cloning.

For expression of the DNA sequence of interest in mammalian host cells,a variety of regulatory regions can be used, for example, the SV40 earlyand late promoters, the cytomegalovirus (CMV) immediate early promoter,and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter.Inducible promoters that may be useful in mammalian cells include butare not limited to those associated with the metallothionein II gene,mouse mammary tumor virus glucocorticoid responsive long terminalrepeats (MMTV-LTR), β-interferon gene, and hsp70 gene [Williams et al.,Cancer Res. 49 (1989) 2735-42; Taylor et al., Mol. Cell. Biol., 10(1990) 165-75]. It may be advantageous to use heat shock promoters orstress promoters to drive expression of the DNA sequence of interest inrecombinant host cells.

In addition, the expression vector may contain a selectable orscreenable marker gene for initially isolating, identifying or trackinghost cells that contain the vector. A number of selection systems may beused for mammalian cells, including but not limited to the Herpessimplex virus thymidine kinase [Wigler et al., Cell 11 (1977) 223],hypoxanthine-guanine phosphoribosyltransferase [Szybalski and Szybalski,Proc. Natl. Acad. Sci. USA 48 (1962) 2026], and adeninephosphoribosyltransferase [Lowy et al., Cell 22 (1980) 817] genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordihydrofolate reductase (dhfr), which confers resistance to methotrexate[Wigler et al., Natl, Acad. Sci. USA 77 (1980) 3567; O'Hare et al.,Proc. Natl. Acad. Sci. USA 78 (1981) 1527]; gpt, which confersresistance to mycophenolic acid [Mulligan & Berg, Proc. Natl. Acad. Sci.USA 78 (1981) 2072]; neomycin phosphotransferase (neo), which confersresistance to the aminoglycoside G-418 [Colberre-Garapin et al., J. Mol.Biol. 150 (1981) 1]; and hygromycin phosphotransferase (hyg), whichconfers resistance to hygromycin [Santerre et al., 1984, Gene 30 (1984)147]. Other selectable markers, such as but not limited to histidinoland Zeocin® can also be used.

5.4.2 Expression Systems and Host Cells

For use with the methods of the invention, the host cell and/or the hostorganism preferably does not express the Bovine Papilloma Virus E1protein. Furthermore, preferably the vector of the invention does notencode the Bovine Papilloma Virus E1 protein.

Preferred mammalian host cells include but are not limited to thosederived from humans, monkeys and rodents, (see, for example, Kriegler M.in “Gene Transfer and Expression: A Laboratory Manual”, New York,Freeman & Co. 1990), such as monkey kidney cell line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293, 293-EBNA, or293 cells subcloned for growth in suspension culture, Graham et al., J.Gen. Virol., 36 (1977) 59; baby hamster kidney cells (BHK, ATCC CCL 10);chinese hamster ovary-cells-DHFR [CHO, Urlaub and Chasin. Proc. Natl.Acad. Sci. 77 (1980) 4216]; mouse sertoli cells [Mather, Biol. Reprod.23 (1980) 243-251]; mouse fibroblast cells (NIH-3T3), monkey kidneycells (CVI ATCC CCL 70); african green monkey kidney cells (VERO-76,ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2);canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human livercells (Hep G2, HB 8065); and mouse mammary tumor cells (MMT 060562, ATCCCCL51).

The vectors of the invention may be synthesized and assembled from knownDNA sequences by well-known techniques in the art. The regulatoryregions and enhancer elements can be of a variety of origins, bothnatural and synthetic. Some host cells may be obtained commercially.

The vectors of the invention containing a DNA sequence of interest canbe introduced into the host cell by a variety of techniques known in theart, including but not limited to, for prokaryotic cells, bacterialtransformation (Hanahan, 1985, in DNA Cloning, A Practical Approach,1:109-136), and for eukaryotic cells, calcium phosphate mediatedtransfection [Wigler et al., Cell 11 (1977) 223-232], liposome-mediatedtransfection [Schaefer-Ridder et al., Science 215 (1982) 166-168],electroporation [Wolff et al., Proc Natl Acad Sci 84 (1987) 3344], andmicroinjection [Cappechi, Cell 22 (1980) 479-4889].

In a specific embodiment, cell lines that express the DNA sequence ofthe invention may be engineered by using a vector that contains aselectable marker. By way of example but not limitation, following theintroduction of the vector, engineered cells may be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the vector confers resistance to theselection and optimally allows only cells that contain the vector withthe selectable marker to grow in culture.

5.4.3 Vaccine Approaches

In certain embodiments, a vector of the invention comprising anexpression cassette of a DNA sequence of interest is administered to asubject to induce an immune response. Specifically, the DNA sequence ofinterest encodes a protein (for example, a peptide or polypeptide),which induces a specific immune response upon its expression. Examplesof such proteins are discussed in section 5.3.

For the delivery of a vector of the invention for use as a vaccine,methods may be selected from among those known in the art and/ordescribed in section 5.4.6.

5.4.4 Gene Therapy Approaches

In a specific embodiment, a vector of the invention comprising anexpression cassette comprising DNA sequences of interest is administeredto treat, or prevent various diseases. The DNA sequence of interest mayencode a protein and/or a biologically active RNA molecule. Gene therapyrefers to therapy performed by the administration to a subject of anexpressed or expressible DNA sequence. In this embodiment of theinvention, the DNA sequences produce their encoded protein or RNAmolecule that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the method of gene therapy, see, Goldspiel etal., Clinical Pharmacy 12 (1993) 488-505; Wu and Wu, Biotherapy 3 (1991)87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32 (1993) 573-596;Mulligan, Science 260 (1993) 926-932; Morgan and Anderson, Ann. Rev.Biochem. 62 (1993) 191-217; May, TIBTECH 1, I (5) (1993) 155-215.Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

The following animal regulatory regions, which exhibit tissuespecificity and have been utilized in transgenic animals, can be usedfor expression of the DNA sequence of interest in a particular tissuetype: elastase I gene control region which is active in pancreaticacinar cells [Swift et al., Cell 38 (1984) 639-646; Ornitz et al., ColdSpring Harbor Symp. Quant. Biol. 50 (1986) 399-409; MacDonald,Hepatology 7 (1987) 425-515]; insulin gene control region which isactive in pancreatic beta cells [Hanahan, Nature 315 (1985) 115-122],immunoglobulin gene control region which is active in lymphoid cells[Grosschedl et al., Cell 38 (1984) 647-658; Adames et al., Nature 318(1985) 533-538; Alexander et al., Mol. Cell. Biol. 7 (1987) 1436-1444],mouse mammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells [Leder et al., Cell 45 (1986) 485-495],albumin gene control region which is active in the liver [Pinkert etal., Genes and Devel. 1 (1987) 268-276], alpha-fetoprotein gene controlregion which is active in the liver [Krumlauf et al., Mol. Cell. Biol. 5(1985) 1639-1648; Hammer et al., Science 235 (1987) 53-58; alpha1-antitrypsin gene control region which is active in the liver [Kelseyet al., Genes and Devel. 1 (1987) 161-171], beta-globin gene controlregion which is active in myeloid cells [Mogram et al., Nature 315(1985) 338-340; Kollias et al., Cell 46 (1986) 89-94]; myelin basicprotein gene control region which is active in oligodendrocyte cells inthe brain [Readhead et al., Cell 48 (1987) 703-712]; myosin lightchain-2 gene control region which is active in skeletal muscle [Sani,Nature 314 (1985) 283-286], and gonadotropic releasing hormone genecontrol region which is active in the hypothalamus [Mason et al.,Science 234 (1986) 1372-1378].

Methods of delivery for gene therapy approaches are well known in theart and/or described in section 5.4.6.

5.4.5 Inhibitory Antisense and Ribozyme

In certain embodiments of the invention a vector of the inventioncontains a DNA sequence of interest that encodes an antisense orribozyme RNA molecule. Techniques for the production and use of suchmolecules are well known to those of skill in the art.

Antisense RNA molecules act to directly block the translation of mRNA byhybridizing to targeted mRNA and preventing protein translation.Antisense approaches involve the design of oligonucleotides that arecomplementary to a target gene mRNA. The antisense oligonucleotides willbind to the complementary target gene mRNA transcripts and preventtranslation. Absolute complementarity, although preferred, is notrequired.

A sequence “complementary” to a portion of an RNA, as referred toherein, means a sequence having sufficient complementarity to be able tohybridize with at least the non-polyA portion of an RNA, forming astable duplex; in the case of double-stranded antisense nucleic acids, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acid.Generally, the longer the hybridizing nucleic acid, the more basemismatches with an RNA it may contain and still form a stable duplex (ortriplex, as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

Antisense nucleic acids should be at least six nucleotides in length,and are preferably oligonucleotides ranging from 6 to about 50nucleotides in length. In specific aspects the oligonucleotide is atleast 10 nucleotides, at least 17 nucleotides, at least 25 nucleotidesor at least 50 nucleotides. In other embodiments of the invention, theantisense nucleic acids are at least 100, at least 250, at least 500,and at least 1000 nucleotides in length.

Regardless of the choice of target sequence, it is preferred that invitro studies are first performed to quantitate the ability of theantisense oligonucleotide to inhibit gene expression. It is preferredthat these studies utilize controls that distinguish between antisensegene inhibition and nonspecific biological effects of oligonucleotides.It is also preferred that these studies compare levels of the target RNAor protein with that of an internal control RNA or protein.Additionally, it is envisioned that results obtained using the antisenseDNA sequence are compared with those obtained using a control DNAsequence. It is preferred that the control DNA sequence is ofapproximately the same length as the test oligonucleotide and that theDNA sequence of the oligonucleotide differs from the antisense sequenceno more than is necessary to prevent specific hybridization to thetarget sequence.

While antisense DNA sequences complementary to the target gene codingregion sequence could be used, those complementary to the transcribed,untranslated region are most preferred.

For expression of the biologically active RNA, e.g., an antisense RNAmolecule, from the vector of the invention the DNA sequence encoding thebio logically active RNA molecule is operatively linked to a strong polIII or pol II promoter. The use of such a construct to transfect targetcells in the patient will result in the transcription of sufficientamounts of single stranded RNAs that will form complementary base pairswith the endogenous target gene transcripts and thereby preventtranslation of the target gene mRNA. For example, a vector of theinvention can be introduced, e.g., such that it is taken up by a celland directs the transcription of an antisense RNA. Such vectors can beconstructed by recombinant DNA technology methods standard in the art.Expression of the sequence encoding the antisense RNA can be by anypromoter known in the art to act in mammalian, preferably human cells.Such promoters can be inducible or constitutive. Such promoters includebut are not limited to: the SV40 early promoter region [Bernoist andChambon, Nature 290 (1981) 304-310], the promoter contained in the 3long terminal repeat of Rous sarcoma virus [Yamamoto, et al., Cell 22(1980) 787-797], the herpes thymidine kinase promoter [Wagner, et al.,Proc. Natl. Acad. Sci. U.S.A. 78 (1981) 1441-1445], the regulatorysequences of the metallothionein gene [Brinster, et al., 1982, Nature296 (1982) 39-42], etc.

In certain embodiments of the invention, a vector of the inventioncontains a DNA sequence, which encodes a ribozyme. Ribozyme moleculesdesigned to catalytically cleave target gene mRNA transcripts can alsobe used to prevent translation of a target gene mRNA and, therefore,expression of a target gene product [see, e.g., PCT InternationalPublication WO90/11364, published Oct. 4, 1990; Sarver, et al., Science247 (1990) 1222-1225].

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. [For a review, see Rossi, Current Biology 4 (1994)469-471]. The mechanism of ribozyme action involves sequence specifichybridization of the ribozyme molecule to complementary target RNA,followed by an endonucleolytic cleavage event. The composition ofribozyme molecules must include one or more sequences complementary tothe target gene mRNA, and must include the well known catalytic sequenceresponsible for mRNA cleavage. For this sequence, see, e.g., U.S. Pat.No. 5,093,246, which is incorporated herein by reference in itsentirety.

While ribozymes that cleave mRNA at site-specific recognition sequencescan be used to destroy target gene mRNAs, the use of hammerheadribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. The sole requirement is that the target mRNA have thefollowing sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Myers, 1995, Molecular Biology andBiotechnology: A Comprehensive Desk Reference, VCH Publishers, New York,(see especially FIG. 4, page 833) and in Haseloff & Gerlach, Nature, 3341988) 585-591, which is incorporated herein by reference in itsentirety.

Preferably the ribozyme is engineered so that the cleavage recognitionsite is located near the 5′ end of the target gene mRNA, i.e., toincrease efficiency and minimize the intracellular accumulation ofnon-functional mRNA transcripts.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onethat occurs naturally in Tetrahymena thermophila (known as the IVS, orL-19 IVS RNA) and that has been extensively described by Thomas Cech andcollaborators [Zaug, et al., Science, 224 (1984) 574-578; Zaug and Cech,Science, 231 (1986) 470-475; Zaug, et al., Nature, 324 (1986) 429-433;published International patent application No. WO 88/04300 by UniversityPatents Inc.; Been & Cech, Cell, 47 (1986) 207-216]. The Cech-typeribozymes have an eight base pair active site, which hybridizes to atarget RNA sequence whereafter cleavage of the target RNA takes place.The invention encompasses those Cech-type ribozymes, which target eightbase-pair active site sequences that are present in the target gene.

Expression of a ribozyme can be under the control of a strongconstitutive pol III or pol II promoter, so that transfected cells willproduce sufficient quantities of the ribozyme to destroy endogenoustarget gene messages and inhibit translation. Because ribozymes unlikeantisense molecules, are catalytic, a lower intracellular concentrationis required for efficiency.

In instances wherein the antisense and/or ribozyme molecules describedherein are utilized to inhibit mutant gene expression, it is possiblethat the technique may so efficiently reduce or inhibit the translationof mRNA produced by normal target gene alleles that the possibility mayarise wherein the concentration of normal target gene product presentmay be lower than is necessary for a normal phenotype. In such cases, toensure that substantially normal levels of target gene activity aremaintained, therefore, nucleic acid molecules that encode and expresstarget gene polypeptides exhibiting normal target gene activity may, beintroduced into cells via gene therapy methods such as those described,below, in Section 5.4.4 that do not contain sequences susceptible towhatever antisense, ribozyme, or triple helix treatments are beingutilized. Alternatively, in instances whereby the target gene encodes anextracellular protein, it may be preferable to co-administer normaltarget gene protein in order to maintain the requisite level of targetgene activity.

Methods of administering the ribozyme and antisense RNA molecules arewell known in the art and/or described in section 5.4.6.

5.4.6 Pharmaceutical Formulations and Modes of Administration

In a preferred aspect, a pharmaceutical of the invention comprises asubstantially purified vector of the invention (e.g., substantially freefrom substances that limit its effect or produce undesiredside-effects). The subject to whom the pharmaceutical is administered inthe methods of the invention is preferably an animal, including but notlimited to animals such as cows, pigs, horses, chickens, cats, dogs,etc., and is preferably a mammal, and most preferably a human.

In certain embodiments, the vector of the invention is directlyadministered in vivo, where the DNA sequence of interest is expressed toproduce the encoded product. This can be accomplished by any of numerousmethods known in the art. The vectors of the invention can beadministered so that the nucleic acid sequences become intracellular.The vectors of the invention can be administered by direct injection ofnaked DNA; use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont); coating with lipids or cell-surface receptors ortransfecting agents; encapsulation in microparticles or microcapsules;administration in linkage to a peptide which is known to enter thenucleus; administration in linkage to a ligand subject toreceptor-mediated endocytosis [see, e.g., Wu and Wu, J. Biol. Chem. 262(1987) 4429-4432] (which can be used to target cell types specificallyexpressing the receptors); etc. In a specific embodiment, the compoundor composition can be delivered in a vesicle, in particular a liposome[see Langer, Science 249 (1990) 1527-1533; Treat et al., 1989, inLiposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365;Lopez-Berestein, ibid., pp. 317-327].

In certain embodiments, the vector of the invention is coated withlipids or cell-surface receptors or transfecting agents, or linked to ahomeobox-like peptide which is known to enter the nucleus [see e.g.,Joliot et al., Proc. Natl. Acad. Sci. USA 88 (1991) 1864-1868], etc.

In certain other embodiments, nucleic acid-ligand complexes can beformed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation.

In yet other embodiments, the vector of the invention can be targeted invivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06 180; WO 92/22635;WO92/20316; WO93/14188, and WO 93/20221).

Methods for use with the invention include, but are not limited to,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, epidural, and oral routes. Methods for use with theinvention further include administration by any convenient route, forexample by infusion or bolus injection, by absorption through epithelialor mucocutaneous linings (e.g., oral mucosa, rectal and intestinalmucosa, etc.). In a specific embodiment, it may be desirable toadminister a vector of the invention by injection, by means of acatheter, by means of a suppository, or by means of an implant, saidimplant being of a porous, non-porous, or gelatinous material, includinga membrane, such as a sialastic membrane, or a fiber. Care must be takento use materials to which the vector does not absorb. Administration canbe systemic or local.

In certain embodiments, a vector of the invention is administeredtogether with other biologically active agents such as chemotherapeuticagents or agents that augment the immune system.

In yet another embodiment, methods for use with the invention includedelivery via a controlled release system. In one embodiment, a pump maybe used [see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14(1989) 201; Buchwald et al., Surgery 88 (1980) 507; Saudek et al., N.Engl. J. Med. 321 (1989) 574]. In another embodiment, polymericmaterials can be used [see Medical Applications of Controlled Release,1974, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.; ControlledDrug Bioavailability, Drug Product Design and Performance, 1984, Smolenand Ball (eds.), Wiley, New York; Ranger and Peppas, Macromol. Sci. Rev.Macromol. Chem. 23 (1983) 61; see also Levy et al., Science 228 (1985)190; During et al., Ann. Neurol. 25 (1989) 351; Howard et al., J.Neurosurg. 71 (1989) 105].

Other controlled release systems are discussed in the review by Langer,Science 249 (1990) 1527-1533.

Pharmaceutical compositions of the invention comprise a therapeuticallyeffective amount of a vector of the invention, and a suitablepharmaceutical vehicle. In a specific embodiment, the term “suitablepharmaceutical vehicle” means approved by a regulatory agency of theFederal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “vehicle” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such suitable pharmaceutical vehicles can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the nucleic acid or protein of theinvention, preferably in purified form, together with a suitable amountof carrier so as to provide the form for proper administration to thepatient. The formulation should suit the mode of administration.

In a specific embodiment, the pharmaceutical of the invention isformulated in accordance with routine procedures as a pharmaceuticalcomposition adapted for intravenous administration to human beings.Typically, compositions for intravenous administration are solutions insterile isotonic aqueous buffer. Where necessary, the pharmaceutical ofthe invention may also include a solubilizing agent and a localanesthetic such as lignocaine to ease pain at the site of the injection.Generally, the ingredients are supplied either separately or mixedtogether in unit dosage form, for example, as a dry lyophilized powderor water free concentrate in a hermetically sealed container such as anampoule or sachette indicating the quantity of active agent. Where thepharmaceutical of the invention is to be administered by infusion, itcan be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the pharmaceutical of theinvention is administered by injection, an ampoule of sterile water forinjection or saline can be provided so that the ingredients may be mixedprior to administration.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The amount of a vector of the invention, which will be effective in thetreatment or prevention of the indicated disease, can be determined bystandard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the stage of indicated disease, and shouldbe decided according to the judgment of the practitioner and eachpatient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

The present invention may be better understood by reference to thefollowing non-limiting Examples, which are provided as exemplary of theinvention. The following examples are presented in order to more fullyillustrate the preferred embodiments of the invention. They should in noway be construed, however, as limiting the broad scope of the invention.

6 EXAMPLES 6.1. Example 1 Cloning and Analysis of The ExpressionProperties of the Vectors super6 and super6wt

The vector plasmids super6 (FIG. 1) and super6wt were prepared fromprevious generation based gene vaccination vectors VI (FIG. 2) and VIwt,respectively. Vectors VI and VIwt are principally synthetic bacterialplasmids that contain a transposon Tn903 derived kanamycin resistancemarker gene [Oka, A., et al., J Mol Biol 147 (1981) 217-226] and amodified form of pMB1 replicon [Yanisch-Perron, C., et al., Gene 33(1985) 103-119] needed for the propagation in Escherichia coli cells.Vectors VI and VIwt also contain a Cytomegalovirus Immediately EarlyPromoter combined with a HSV1 TK leader sequence and rabbit β-globingene sequences, which both are derived from plasmid pCG [Tanaka, M., etal., 60 (1990) Cell 375-386]. The latter elements are needed forexpressing from the nef coding sequence derived from a HAN2 isolate ofthe HIV-1 strain [Sauermann, U., et al., AIDS Research. Hum. Retrov. 6(1990) 813-823]. The expression vectors for the Nef carry clustered tenhigh affinity E2 binding sites (derived from plasmid pUC1910BS,unpublished) just upstream of the CMV promoter.

The parent vector VI contains a modified E2 coding sequence: the hingeregion of E2 (amino acids 192-311) is replaced with four glycine-alaninerepeats from EBNA1 protein of Epstein-Barr Virus [Baer, R. J., et al.,Nature 310 (1984) 207-211]. The protein encoded by this sequence wasnamed as E2d192-311+4G, The parent vector VIwt contains an expressioncassette for wild type E2 protein of the bovine papilloma virus type 1with point mutations introduced for the elimination E3 and E4 openreading frame (ORF) coding capacity by two stop codons into both theseORFs. In the vectors the E2 coding sequences are cloned between a Roussarcoma virus proviral 5′ LTR [Long, E. O., et al., Hum. Immunol. 31(1991) 229-235] and bovine growth hormone polyadenylation region[Chesnut, J. D., et al., J Immunol Methods 193 (1996) 17-27].

Plasmid vectors super6 and super6wt were constructed by deleting fromthe respective parent vectors VI and VIwt all beta-globin sequencesdownstream of the nef gene except the second intron of the rabbitbeta-globin gene. The beta-globin sequences (especially the fragment ofthe exon) show some homology with sequences in the human beta-globingene, whereas the intron lacks any significant homology to human genomicsequences. The intron was amplified by PCR from the plasmid pCG [Tanaka,M. et al., Cell 60 (1990) 375-386] using oligonucleotides with somemismatches for modifying the sequences of splicing donor and acceptorsites of the intron to the perfect match to consensus motifs. The HerpesSimplex Virus type 1 thymidine kinase gene polyadenylation region frompHook [Chesnut, J. D., et al., J Immunol Methods 193 (1996) 17-27] wasthen cloned just next to the 3′-end of the intron, because in parentplasmids the rabbit β-globin polyadenylation signal were used.

The expression properties of the Nef and E2 proteins expressed by theplasmid vectors super6 and super6wt were analyzed and compared with theexpression properties of the Nef and E2 proteins expressed by VI andVIwt by Western blotting [Towbin et al., Proc Natl Acad Sci USA 76(1979) 4350-4354] with monoclonal antibodies against Nef and E2. First,Jurkat cells (a human T-cell lymphoblast cell line) were transfected byelectroporation [Ustav et al. EMBO J 2 (1991) 449-457] with 1 μg ofsuper6, super6WT or equimolar amounts of the plasmids VI, VIwt. As acontrol an equimolar amount of vector II (FIG. 3), which contains anidentical Nef cassette but no E2 coding sequence, was used. Carrier DNAwas used as a negative control. Briefly, the plasmid and carrier DNAwere mixed with the cell suspension in a 0.4 cm electroporation cuvette(BioRad Laboratories, Hercules, USA) followed an electric pulse (200V; 1mF) using Gene Pulser IITM with capacitance extender (BioRadLaboratories, Hercules, USA).

Forty-nine hours post-transfection the cells were lysed by treating witha sample buffer containing 50 mM Tris-HCl pH 6.8; 2% SDS, 0.1%bromophenol blue, 100 mM dithiothreitol, and 10% (v/v) glycerol. Thelysates were run on a 10% or 12.5% SDS-polyacrylamide gel andsubsequently transferred onto a 0.45 μm PVDF nitrocellulose membrane(Millipore). The membrane was first blocked overnight with a blockingsolution containing 5% dry milk (fat-free), 0.1% Tween 20 in 50 mMTris-HCl pH 7.5; 150 mM NaCl and thereafter incubated for 1 h withdiluted monoclonal anti-Nef antisera (1:100) or anti-E2-antisera(1:1000) in the blocking solution. After each incubation step, unboundproteins were removed by washing strips three times with TBS-0.1%Tween-20. The binding of primary immunoglobulins was detected byincubating the strips with horse raddish peroxidase conjugate anti-mouseIgG (Labas, Estonia) followed by visualization using a chemoluminesencedetection system (Amersham Pharmacia Biotech, United Kingdom).

The results are shown in FIG. 4. The expression of the Nef protein isshown on panel A and the expression of E2 protein on panel B. The arrowsindicate the right molecular sizes of the Nef and E2 proteins. Theexpression level of the E2d192-311+4GA is very low and for this reasoncannot be seen on the blot presented in FIG. 4.

The amounts of Nef expressed from the plasmids super6, super6wt, VI andVIwt (lanes 1-4 in FIG. 4A) are quite similar (FIG. 4, panel A, lanes 1to 4). Much less protein is produced from plasmid II (lane 5). Theexpression levels of the Nef protein are higher from vectors containingwtE2 (cf. lane 1 compared with lane 2 and lane 3 compared with lane 4).This is in accordance with the expression levels of E2 andE2d192-311+4GA proteins from these plasmids (FIG. 4, panel B).

6.2. Example 2 Cloning and Analysis of the Expression Properties ofPlasmids in Series product1 and NNV

To increase the copy number of the vectors super 6 and super6wt inEscherichia coli further modifications were made in these vectors. TheTn903 kanamycin resistance gene, pMB1 replicon and ten E2 binding siteswere removed by HindIII/NheI digestion followed by replacing with theHind III/NheI fragment from retroviral vector pBabe Neo [Morgenstern, J.P. and Land, H., Nucleic Acids Research 18 (1990) 3587-3596]. Thisfragment contains a modified pMB1 replicon and the Tn5 kanamycinresistance gene that allow relaxed high copy-number replication of theplasmids in bacteria. The new plasmids were named as the product1 (FIG.5), and product1wt respectively. An unsuccessful attempt to reinsert theten E2 binding sites back into the blunted NheI site upstream of the CMVpromoter of the product1 resulted in vector New Vector NNV,respectively, with only two binding sites integrated in the plasmid.

Additional ten E2 binding sites were inserted from plasmid pUC1910BSinto the New Vector in just downstream the E2 expression cassette. Thesenew vectors were named NNV-1 and NNV-2 (FIG. 6A). For replacing theE2d192-311+4GA with wt E2 (with deleted E3 and E4 ORFs), theE2d192-311+4GA coding sequence containing Bsp120I fragment was replacedwith wtE2 containing an analogous Bsp120I fragment from the super6wt.Generated plasmids were named NNV-1wt and NNV-2wt (FIG. 6B),respectively. The numbers 1 or 2 in vectors of the NNV series mark theorientation of the 10 E2 binding sites region relative to the E2expression cassette.

The expression properties of the Nef protein from the NNV plasmids, i.e.NNV-1, NNV-2, NNVwt and NNV-2wt, after the transfection of Jurkat cellsby electroporation at a concentration of 1 ìg of the plasmid wereanalyzed and compared with the expression properties of the Nef proteinsfrom super6 and super6 wt by Western blotting essentially as describedin Example 1. The amounts of super6 and super6wt used for thetransfection were 0.95 and 1 ìg, respectively. The results are shown inFIG. 7.

NNV-1 and NNV-2 vectors have expression potential similar to plasmidsuper6 as evident from the comparison of lanes 1 and 2 on FIG. 8 withlane 5. The same applies to vectors NNV-1wt, NNV-2wt and super6wt(compare lanes 3 and 4 with lane 6 on FIG. 7). In accordance with theprevious results the plasmids expressing wt E2 produce more Nef proteinthan E2d192-311+4GA vectors do (compare lane 1 with lane 3 and lane 2with lane 4 in FIG. 7). In view of this and since the Nef expressionfrom NNV-2wt was slightly higher than that from NNV-1wt, vector NNV-2wtwas selected for further tests.

6.3 Example 3 Analysis of the expression properties of NNV-2wt

To analyse the expression properties of NNV-2wt, four different celllines, i.e. the Jurkat (human T-cell lymphoblasts), P815 (mousemastocytoma cells), CHO (Chinese Hamster Ovary cells) lines and RD(human embryo rhabdomyosarcoma cells), were transfected byelectroporation and analyzed for their expression of Nef of and E2. Toreveal the transcription activation and maintenance properties mediatedby E2 protein and E2 oligomerized binding sites product1wt, which lacksthe E2 binding sites (FIG. 5), was used as a control. An additionalcontrol plasmid was plasmid NNV-2wtFS, which differs from NNV-2wt bycontaining a frameshift introduced into E2 coding sequence, whereby itdoes not express functional E2 protein.

Each cell line was transfected with different amounts of the vector DNAby electroporation essentially as described in Example 1. Time-pointswere taken approximately two and five days after transfection. Theresults of analyses are presented in FIGS. 8 to 10.

The Jurkat cells were transfected with 0.5 μg or 2 μg of the NNV-2wt(lanes 1, 2, 8, and 9 in FIG. 8) and equal amounts of the plasmidsNNV-2wtFS (lanes 3, 4, 10, and 11 in FIG. 8) and product1wt (lanes 5, 6,12, and 13 in FIG. 8) or carrier only (lanes 7 and 14 in FIG. 8).Time-points were taken 44 hours (lanes 1-7) and 114 hours (lanes 8-14)after transfection: The expression of the Nef and E2 proteins wasanalyzed by Western blotting essentially as described in Example 1.

The P815 cells were transfected with 0.5 μg or 2 μg of the NNV-2wt(lanes 1, 2, 8, and 9 in FIG. 9) and equal amounts of the plasmidsNNV-2wtFS (lanes 3, 4, 10, and 11 in FIG. 9) and product1wt (lanes 5, 6,12, and 13 in FIG. 9) or carrier only (lanes 7 and 14 in FIG. 9).Time-points were taken 45 hours (lanes 1-7) and 119 hours (lanes 8-14)after transfection: The expression of the Nef proteins was analyzed byWestern blotting essentially as described in Example 1. The blot withanti-E2 antibodies 119 h post-transfection is not shown, because nospecial signal could be detected. Generally, the expression level of theNef protein correlated with the expression level of E2 protein in thesecells, which confirms the fact that the function of the E2 protein is toactivate the transcription and to help the plasmid to be maintained fora longer time in the proliferating cells.

The CHO cells were transfected with 0.5 μg or 2 μg of the NNV-2wt (lanes1, 2, 8, and 9 in FIG. 10) and equal amounts of the plasmids NNV-2wtFS(lanes 3, 4, 10, and 11 in FIG. 10) and product1wt (lanes 5, 6, 12, and13 in FIG. 10) or carrier only (lanes 7 and 14 in FIG. 10). Time-pointswere taken 48 hours (lanes 1-7) and 114 hours (lanes 8-14) aftertransfection. The expression of the Nef and E2 proteins was analyzed byWestern blotting essentially as described in Example 1.

The RD cells were transfected with 0.5 μg or 2 μg of the NNV-2wt (lanes1, 2, 8, and 9 in FIG. 11) and equal amounts of the plasmids NNV-2wtFS(lanes 3, 4, 10, and 11 in FIG. 11) and product1wt (lanes 5, 6, 12, and13 in FIG. 11) or carrier only (lanes 7 and 14 in FIG. 11) Time-pointswere taken 39 hours (lanes 1-7) and 110 hours (lanes 8-14) aftertransfection. The expression of the Nef protein was analyzed by Westernblotting essentially as described in Example 1.

In all four cell lines the expression level of the Nef protein, taken atearlier time points (lanes 1-7 in FIGS. 8-11) and at later time points(lanes 8-14 in FIGS. 8-11) hours, from the NNV-2wt was higher than fromcontrol vectors. The superiority of the NNV-2wt was more obvious atlater time-points as evident from the comparison of lane 8 with lanes 10and 12 in FIG. 8, and also from the comparison of lane 9 with lanes 11and 13 in FIGS. 8, 9 and 10.

The expression pattern of RNA from these plasmids was also analyzedusing the Northern analysis [Alwine, J. C, et al., Proc Natl Acad SciUSA 74 (1977) 5350-5354] for the NNV-2wt vector. For this purpose,Jurkat and CHO cells were transfected with 2 μg of the NNV-2wt. For thetransfection of P815 cells 10 μg of NNV-2wt were used. The transfectionswere made essentially as described in Example 1. Forty-eight hourspost-transfection total RNA was extracted using RNAeasy kit (Qiagen) andsamples containing 21 μg (P815), 15 μg (CHO) or 10 μg (Jurkat) of theRNA were analysed by electrophoresis under the denaturing conditions(1.3% agarose gel containing 20 mM MOPS pH 7.0; 2 mM NaOAc; 1 mM EDTA pH8.0; 2.2M formaldehyde). The running buffer contained the samecomponents except formaldehyde. The samples were loaded in a buffercontaining formamide and formaldehyde. After the electrophoresis theseparated RNAs were blotted onto the HybondN+ membrane (AmershamPharmacia Biotech, United Kingdom) and hybridization with aradio-labeled nef coding sequence, E2 coding sequence or whole vectorprobes was carried out. The RNA from cells transfected with the carrierwas used as a control. The results of the Northern blot analyses areshown in FIG. 12.

The results indicate that no other RNA species than complementary mRNAsfor E2 and nef are expressed from the vector, since no additionalsignals can be detected with the whole vector probe compared with nefand E2 specific hybridizations (compare lanes 1-12 with lanes 13-18 inFIG. 12).

6.4 Example 4 Analysis of the Attachment of the NNV-2wt to MitoticChromosomes

The attachment of the NNV-2wt to mitotic chromosomes in CHO cells wasanalyzed by fluoresence in situ hybridisation (FISH) [Tucker J. D., etal., In: J. E. Celis (ed.), Cell Biology: A Laboratory Handbook, vol 2,p. 450-458. Academic Press, Inc. New York, N.Y. 1994.].

Thirty-six hours post-transfection the CHO cells by electroporation with1 μg of NNV-2wt or with equimolar amounts of the control plasmidsNNV-2wtFS and product1wt (performed essentially as described inExample 1) the cultures were treated with colchicin (Gibco) forarresting the cells in metaphase of the mitosis. Briefly, cells wereexposed to colchicine added to medium at final concentration of 0.1μg/ml for 1-4 h to block the cell cycle at mitosis. Blocked cells wereharvested by a trypsin treatment and suspended in a 0.075M KCl solution,incubated at room temperature for 15 min, and fixed in ice-coldmethanol-glacial acetic acid (3:1, vol/vol). The spread-out chromosomesat metaphase and nuclei at interphase for fluorescence in situhybridization analyses were prepared by dropping the cell suspension onwet slides. Several slides from one culture were prepared.

Hybridization probes were generated by nick-translation, usingbiotin-16-dUTP as a label and plasmid Product1wt as template. A typicalnick-translation reaction mixture contained a nick-translation buffer,unlabeled dNTPs, biotin-16-dUTP, and E. coli DNA polymerase.

Chromosome preparations were denatured at 70° C. in 70% formamide (pH7.0-7.3) for 5 min, then immediately dehydrated in a series of washes(70%, 80%, and 96% ice-cold ethanol washes for 3 min each), andair-dried. The hybridization mixture (18 μl per slide) was composed of50% formamide in 2×SSC (1×SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate), 10% dextran sulfate, 150 ng of biotinylated plasmid probe DNAand 10 μg of herring sperm carrier DNA. After 5 min of denaturation at70° C., probe DNA was applied to each slide, sealed under a coverslip,and hybridized for overnight at 37° C. in a moist chamber. The slideswere washed with three changes of 2×SSC, nd 2×SSC containing 0.1% IGEPALCA-630 (Sigma Chemical Co.) at 45° C. Prior to the immunofluorescencedetection, slides were preincubated for 5 min in PNM a buffer [PN buffer(25.2 g Na₂HPO₄.7H₂O, 083 g NaH₂PO₄._□ H₂O and 0.6 ml of IGEPAL CA-630in 1 μliter of H₂O] with 5% nonfat dried milk and 0.02% sodium azide).

Subsequently, the probe was detected with fluorescein isothiocyanate(FITC)-conjugated extravidin. The signal was amplified with biotinylatedantiavidin antibody and a second round of extravidin-FITC treatment.Between each of the steps, the slides were washed in PN buffercontaining 0.05% IGEPAL CA-630 at room temperature for 2×5 min.Chromosomes were counterstained with propidium iodide and mounted inp-phenylenediamine antifade mounting medium.

Slides were analyzed with a Olympus VANOX-S fluorescence microscopeequipped with appropriate filter set.

The results are shown in Table 1.

TABLE 1 Chromosomal attachment of the NNV-2wt. Metaphases with episomalsignal Analyzed Culture on chromosomes metaphases % 0.5 μg 11 158 7NNV-2wt 0.5 μg 0 100 0 NNV-2wtFS 0.48 μg 0 100 0 product1wt carrier 0100 0

The data indicate clearly that the E2 protein and its binding sites areneeded for the chromosomal attachment because only the NNV-2wt but nottwo other vectors have this ability.

6.5 Example 5 Stability of NNV-2wt During Propagation in Bacterial Cells

The stability of NNV-2wt during propagation in bacterial cells wastested. The plasmid NNV-2wt was mixed with competent Escherichia colicells of the DH5alpha strain [prepared as described in Inoue, H., etal., Gene 96 (1990) 23-28] and incubated on ice for 30 minutes.Subsequently, the cell suspension was subjected to a heat-shock for 3minutes at 37° C. followed by a rapid cooling on ice. One milliliter ofLB medium was added to the sample and the mixture was incubated for 45minutes at 37° C. with vigorous shaking. Finally, a portion of the cellswas plated onto dishes containing LB medium with 50 μg/ml of kanamycin.On the next day, the cells from a single colony were transferred ontothe new dishes containing the same medium. This procedure was repeateduntil four generations of bacteria had been grown, and the plasmid DNAfrom the colonies of each generation was analyzed.

One colony from each generation was used for an inoculation of 2 ml LBmedium containing 50 μg/ml of kanamycin followed by an overnightincubation at 37° C. with vigorous shaking. The cells were harvested andthe plasmid DNA was extracted from the cell using classical lysis byboiling. [Sambrook, S., et al., Molecular Cloning A Laboratory Manual.Second ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.]. The samples were digested with restriction endonuclease XbaI(Fermentas, Lithuania) and analyzed by agarose gel electrophoresis incomparison with the original DNA used for transformation. The resultsare shown in FIG. 13.

As can be seen in FIG. 13, the vector is stable during the passage inEscherichia coli cells: no colonies with re-arrangements were observedwhen compared with the DNA used for transformation (lane 9).

6.6 Example 6 Stability of NNV-2wt in Eukaryotic Cells

The stability of the plasmid NNV-2wt as a non-replicating episomalelement was also analyzed in eukaryotic cells. For this purpose the CHOand Jurkat cells were transfected with 2 μg of NNV-2wt. Total DNAs ofthe cells were extracted at 24, 72 or 96 hours post-transfection.Briefly, the cells were lyzed in 20 mM Tris-HCl pH 8.0; 10 mM EDTA pH8.0; 100 mM NaCl; 0.2% SDS; in presence of 200 μg/ml of proteinase K(Fermentas, Lithuania). Next, the samples were extracted sequentiallywith phenol and with chloroform and precipitated with ethanol. Thenucleic acids were resuspended in 10 mM Tris-HCl pH 8.0; 1 mM EDTA pH8.0; 20 μg/ml of RNase A (Fermentas, Lithuania) and incubated for 1 hourat 37° C. Finally the DNA was re-precipitated with ammonium acetate andethanol, washed with 70% ethanol and resuspended in 10 mM Tris-HCl pH8.0; 1 mM EDTA pH 8.0. The samples were digested with differentrestriction endonucleases: with Eco81 (Fermentas, Lithuania) that hastwo recognition sites on the plasmid, with HindIII (Fermentas,Lithuania) that does not cut the NNV-2wt DNA and with DpnI (New EnglandBiolabs, USA) that digest only DNA synthesized in Escherichia colicells. Restricted DNAs were separated on TAE agarose electrophoresis andanalyzed by Southern blotting [Southern, E. M. J. Mol. Biol. 98 (1975)503-517] with a vector specific radiolabeled probe. The results areillustrated on FIG. 14. As obvious from comparison of the fragment sizesof Eco81I digestion (lanes 1, 2 and 7 in FIG. 14) with respective markerlanes no arrangements of the vector were detected in the assay. Neitherwere signals observed at a position different from the marker lanes incases of the Hind III (lanes 3, 4 and 8 in FIG. 14) or HindIII/DpnI(lanes 5, 6 and 9 in FIG. 14) digestion indicating that integrationand/or replication events were not observed.

6.7 Example 7 Analysis of Replication of the NNV-2wt in the Presence ofHuman Papillomaviral Replication Factors

It has been demonstrated previously that papillomaviral proteins areable to initiate the replication of heterologous ori-containing plasmidsfrom many other human and animal papillomaviruses [Chiang, C. M., etal., Proc Natl Acad Sci USA 89 (1992) 5799-5803]. Although NNV-2wt doesnot contain an intact viral origin of replication, it was tested how thereplication is initiated in the presence of human papillomavirus type 1E1 and E2 proteins. CHO cells were transfected with one microgram ofeither plasmids NNV-2wt, NNV-2wtFS or product 1 alone or with 4.5 μg ofthe HPV-11 E1 expression vector pMT/E1 HPV11 or with same amount ofpMT/E1 HPV11 and 4.5 μg HPV-11 E2 protein expression vector pMT/E2 HPV11 as indicated on the top of the FIG. 15. Transfections were doneessentially as described in Example 1. E1 and E2 expression vectors aredescribed previously (Chiang, C. M. et al., supra). An equimolar amountof HPV-11 replication origin containing plasmid HPV11ORI was transfectedwith the same expression vectors as a positive control.

Low-molecular weight DNA was extracted by modified Hirt lysis [Ustav, etal., EMBO J 2 (1991) 449-457] at 67 hours post-transfection. Briefly,the cells washed with PBS were lyzed on ice at 5 minutes by addingalkaline lysis solutions I (50 mM glucose; 25 mM Tris-HCl, pH 8.0; 10 mMEDTA, pH 8.0) and II (0.2M NaOH; 1% SDS) in a ratio of 1:2 onto thedishes. The lysates were neutralized by 0.5 vol solution III (a mixtureof potassium acetate and acetic acid, 3M with respect to potassium and5M with respect to acetate). After centrifugation the supernatant wasprecipitated with isopropanol, resuspended and incubated at 55° C. in 20mM Tris-HCl pH 8.0; 10 mM EDTA pH 8.0; 100 mM NaCl; 0.2% SDS; inpresence of 200 μg/ml of proteinase K (Fermentas, Lithuania). Next, thesamples were extracted sequentially with phenol and with chloroformfollowed by precipitation with ethanol. The nucleic acids wereresuspended in 10 mM Tris-HCl pH 8.0; 1 mM EDTA pH 8.0; 20 μg/ml RNase A(Fermentas, Lithuania) and incubated for 30 min at 65° C. The sampleswere digested with linearizing endonuclease (NdeI; Fermentas, Lithuania)in case of the vectors or HindIII (Fermentas, Lithuania) in case of theHPV11ORI) and DpnI (New England Biolabs, USA) (breaks non-replicatedDNA), followed by Southern blotting performed essentially as describedearlier using a vector specific radiolabeled probe. For positive controlof hybridization appropriate markers of the linearized vectors andHPV11ORI were used (lanes marked as M on FIG. 15). As seen from theresults set forth in FIG. 15, no replication signal was detected in caseof any vector plasmids.

6.8 Example 8 Analysis of the E2 and its Binding Sites DependentSegregation Function of the Vectors in Dividing Cells

As has been described previously, bovine papillomavirus type 1 E2protein in trans and its multiple binding sites in cis are bothnecessary and sufficient for the chromatin attachment of the episomalgenetic elements. The phenomenon is suggested to provide a mechanism forpartitioning viral genome during viral infection in the dividing cells[Ilves, I., et al., J. Virol. 73 (1999) 4404-4412]. Because bothfunctional elements are also included into our vector system, the aim ofthis study was analyze the importance of the E2 protein and oligomerizedbinding sites for maintenance of the transcriptionally active vectorelement in population of dividing cells.

For this purpose the Nef coding sequence of the vectors NNV-2wt andsuper6wt was replaced with coding sequence of the destabilized form ofgreen fluorescent protein (d1EGFP) derived from vector pd1EGFP-N1(Clontech Laboratories). Because the half-life of this protein is asshort as 1 hour, it does not accumulate in the cells and the d1EGFPexpression detected by flow cytometer correlates with the presence oftranscriptionally active vector in these cells.

From NNV-2wt the nef coding sequence was removed and SmaI-NotI fragmentfrom the pd1EGFP-N1 was inserted instead of it. New vector was named as2wtd1EGFP (FIG. 16). Similar replacement was made in case of super6 wtfor generation gf10bse2 (FIG. 17), respectively. The recognitionsequence for restrictional endonuclease SpeI was introduced into theEcoRI site in the super6wt just upstream the ten E2BS. The vectorgf10bse2 is derived from this plasmid by replacing the Nef codingsequence containing NdeI-Bst1107I fragment with d1EGFP coding sequencecontaining fragment from 2wtd1EGFP, cut out with same enzymes.

Negative control plasmids lacking either functional E2 coding sequenceor its binding sites were also made: The frameshift was introduced intothe E2 coding sequence in context of the 2wtd1EGFP by replacing E2coding sequence containing Bsp120I-Bsp120I with similar fragment fromplasmid NNV-2wtFS. The resulting vector was named as 2wtd1EGFPFS (FIG.18). For the construction the control plasmid NNVd1EGFP (FIG. 19) thewhole E2 expression cartridge (as well bacterial replicon) from the2wtd1EGFP was removed by Bst1107 and NheI digestion. The replicon wasreconstituted from plasmid product1 as HindIII (filled in)-NheIfragment.

Jurkat cells were transfected by electroporation with 1 μg of the vector2wtd1EGFP or with equimolar amounts of the plasmids 2wtd1EGFPFS,NNVd1EGFP, gf10bse2 or with carrier DNA only as described in Example 1.At different time-points post-transfection the equal aliquots of thecell suspension were collected for analysis and the samples were dilutedthereafter with the fresh medium. At every time-point total number ofthe cells as well the number of the d1EGFP expressing cells were countedby flow cytometer (Becton-Dickinson FACSCalibur System). With thesedata, the percentages of d1EGFP expressing cells, alterations of totalnumbers of cells and numbers of d1EGFP expressing cells in samples werecalculated using the carrier-only transfected cells as a negativecontrol for background fluorescence. The calculations of cell numberswere done in consideration of the dilutions made. Finally, the errorvalues were calculated based on technical data of the cytometer aboutfluctuations of speed of the flow.

Two independent experiments were done. First, the maintenance of d1EGFPexpressed from the plasmids 2wtd1EGFP, 2wtd1EGFPFS and NNVd1EGFP wereanalyzed during the eight days post-transfection. In the secondexperiment the maintenance of d1EGFP expressed from the plasmids2wtd1EGFP, 2wtd1EGFPFS and gf10bse2 were analyzed during the thirteendays post-transfection.

As is obvious from FIGS. 20 and 21, there was no difference of thegrowth speed of the cells transfected with any vector or carrier only.It means that differences in the d1EGFP expression maintenance are notcaused by influences of transfected vectors themselves on the dividingof the cells. Also, during the assay the logarithmic growth of the cellswere detected, except the period until second time-point in theexperiment represented in FIG. 20. This lag period of the growth isprobably caused by the electroporation shock of the cells, because thefirst time-point was taken already 19 hours after the transfection.

As illustrated in FIGS. 22 and 23, the percentages of green fluorescentprotein expressing cells decrease in all populations transfected witheither plasmid, because the vectors do not replicate in the cells.However, as is seen on the charts, the fraction of positive cellsdeclines more rapidly in cases of control vectors, if compared with the2wtd1EGFP or gf10bse2. If compared with each other, the gf10bse2 haveclear benefit to 2wtd1EGFP (FIG. 23.). There is also a notabledifference of maintenance between control plasmids 2wtFSd1EGFP andNNVd1EGFP (FIG. 22).

These differences between the vectors become much more obvious, if thedata are represented as alterations of the numbers of the d1EGFPexpressing cells in the populations (FIGS. 24 and 25). The numbers ofthe positive cells in cases of the control plasmids are not notablechanged during the assay. In contrast, in case of the 2wtd1EGFP thenumber of d1EGFP expressing cells increases during the first week afterthe transfection becoming approximately five to ten times higher than incontrol samples (FIG. 24). After this time-point the number start todecrease (FIG. 25). The difference of maintenance is strongest in thecase of the gf10bse2 vector. The number of positive cells increasescontinuously during the analyses period. After two weeks it is 6 timeshigher than in the sample transfected with 2wtd1EGFP and 45 times higherthan in the population transfected with frameshift mutant (FIG. 25).

The data demonstrate clearly that the vector system of the presentinvention has active mechanism of segregation based on anuclear-anchoring protein, i.e. bovine papillomavirus type 1 E2 proteinand its binding sites that promotes its maintenance in a population ofproliferating cells as a transcriptionally active element.

6.9 Example 9 Cloning of the AIRE Gene into super6wt and Expression inan Epithelial Cell Line

The AIRE gene coding for the AIRE protein (AIRE=autoimmune regulator) ismutated in an autosomally heredited syndrome APECED (Autoimmunepolyendocrinopathy candidiasis ectodermal dystrophy). AIRE is expressedin rare epithelial cells in the medulla of thymus and in the dendriticcells in peripheral blood and in peripheral lymphoid organs. APECEDcould thus be treated by transferring the non-mutated AIRE gene ex vivoto peripheral blood dendritic cells, followed by the introduction of thecorrected dendritic cells back to the patient. To test this possibilityhuman AIRE gene and the homologous murine AIRE gene were transferred toCOS-1 cells.

For cloning of the AIRE gene into Super6wt a maxi-preparation of thevector was prepared. First a transfection with Super6wt was done toTOP10-cells (chemically competent Escherichia coli by Invitrogen)according to manufacturer's protocol. Briefly, the cells were incubatedon ice for 30 minutes, after which a heat shock was performed in a waterbath at +42° C. for 30 seconds. The cells were then transferred directlyon ice for 2 minutes and grown in 250 μl of SOC-medium (2% tryptone,0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20mM glucose) at +37° C. with shaking for 1 hour.

Plating was done on LB-plates using kanamycin (50 μg/ml) for selection.For maxiprep, colonies were transferred to 150 ml of LB-solutioncontaining kanamycin (50 μg/ml) and grown overnight at +37° C. withshaking. Preparation of maxiprep was done using Qiagen's Plasmid MaxiKit according to manufacturer's protocol.

A digestion with BamHI and SalI restriction enzymes was used to checkthe vector. The reaction mixture contained 500 ng of Super6w, 5 U ofBamHI, 5 U of SalI, 2 μl of 2×TANGO buffer (both the restriction enzymesand buffer from Fermentas) and sterile water in total volume of 10 μl.The digestion was carried out at +37° C. for one hour.

The digested vector was checked with 1% agarose gel containing ethidiumbromide 1 μg/ml in 1×TAE-buffer.

For cloning of the PCR amplified AIRE gene and Aire fragments into theSuper6wt, 4 μg of Super6wt was digested with 10 U NotI restrictionenzyme (MBI Fermentas, in 2 μl enzyme buffer and sterile water added toa final volume of 20 μl. The digestion was carried at +37° C. for 1.5hours, after which 1 U of ZIP-enzyme (alkaline phosphatase) was added tothe reaction mixture and incubated further for 30 minutes. TheZIP-enzyme treatment was done to facilitate the insertion of the AIREgene into the vector by preventing the self-ligation of the vector backto a circular mode. After the digestion the vector was purified usingGFXTM PCR DNA and Gel Band Purification Kit (Amersham Pharmacia Biotech)and dissolved in to a concentration of 0.2 micrograms/microliter.

Human and mouse AIRE-gene PCR-products were also digested with NotIrestriction enzyme. To the digestion, 26 μl of PCR product, 3 μl of anappropriate enzyme buffer and 10 U of NotI restriction enzyme (thebuffer and enzyme from MBI Fermentas) was used. The digestion wascarried out at +37° C. for 2 hours, after which digested PCR-productswere purified and dissolved in sterile water to a volume of 10 μl.

The PCR amplified and digested human and mouse AIRE genes were ligatedto Super6wt by a T4 DNA ligase (MBI Fermentas). The digested insert DNAwas taken (a total volume of 10 μl), 1.5 μl of ligase buffer (MBIFermentas), 5 U of T4 DNA ligase and sterile water was added to a finalconcentration of 15 μl. The ligation was carried out at +17° C.overnight.

After the ligation 10 μl of ligation reaction mixture was taken fortransfection into TOP10 cells according to manufacturer's protocol. Thecells were incubated on ice for 30 minutes, after which a heat shock wasperformed in a water bath at +42° C. for 30 seconds. The cells were thentransferred directly on ice for 2 minutes and grown in 250 μl ofSOC-medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10mM MgCl₂, 10 mM MgSO₄, 20 mM glucose) at +37° C. with shaking for 1hour.

The transfected bacterial cells were plated onto LB-kanamycin plates andcolonies were picked on the following day to 2 ml of LB-medium (1%tryptone, 0.5% yeast extract, 170 mM NaCl) with kanamycin and grownovernight at +37° C.

Miniprep DNA preparations from selected colonies were purified usingQiagen's Plasmid Midi Kit and dissolved to a volume of 50 μl of sterilewater. The presence and size of the insert was checked with NotI andBamHI digestion. 10 μl of miniprep DNA was taken for digestion, 5 U ofNotI and 5 U of BamHI enzymes, 2 ml of R+enzyme buffer and sterile waterwas added to a final volume of 20 μl. The digestion was carried out at+37° C. for 1 hour.

The orientation of the insert was analysed with BamHI restrictionenzyme. Ten μl of minprep DNA was taken, 5 U of BamHI, 2 μl of BamHIbuffer (MBI Fermentas) and sterile water was added to a final volume of20 μl. The digestion was carried out for 1 hour at +37° C. and theproducts were checked on a 1% agarose gel with EtBr in 1×TAE.

On the basis on these results, a plasmid containing a mouse AIRE-geneand a plasmid containing a human AIRE-gene were picked and maxiprepswere prepared. Briefly, 0.5 ml of E. coli cell suspension containing theplasmid of interest or a miniprep culture was added to a 150 mlLB-medium containing kanamycin (50 μg/ml) and grown overnight at +37° C.Maxiprep DNAs were prepared using Qiagen's Plasmid Maxi Kit.

The plasmid containing the mouse AIRE-gene was designated as pS6 wtmAIREand plasmid containing the human AIRE-gene as pS6 wthAIRE.

The generated vectors were sequenced for approximately 500 bp from bothends to verify the orientation and correctedness of the insert. Thesequencing was performed using the dideoxy method with PE Biosystem'sBig Dye Terminator RR-mix, which contains the four different terminatingdideoxynucletide triphosphates labeled with different fluorescentlabels.

Plasmids containing the AIRE gene and AIRE gene fragments were insertedinto selected cell lines to check the expression of the protein withWestern blot after the transfection.

Cos-1 cells were harvested with trypsin-EDTA (Bio Whittaker Europe)solution and suspended 10×106 cells/ml into Dulbecco's MEM (LifeTechnologies) medium and 250 μl of cell suspension was taken fortransfection. The transfection of Cos-1 cells was performed usingelectroporation with 2.5×106 cells, 50 μg of salmon sperm DNA as acarrier and 5 μg of appropriate vector. The transfections were made withpS6 wthAiRE, pS6 wtmAIRE, Super6wt, pCAIRE, psiAIRE and pCAIRE S1-4.pCAIRE and psiAIRE are positive human AIRE controls, pCAIRE S1-4 is apositive mouse AIRE control and Super6wt is a negative control.

The electroporation was done using Biorad's Gene Pulser with capacitance960 μFd, 240 V and 1 pulse. After the pulse the cells were kept at roomtemperature for 10 minutes and 400 μl of medium was added. The cellswere transferred to 5 ml of medium and centrifuged for 5 minutes with1000 rpm. Cells were plated and grown for 3 days at +37° C., 5% CO2.

The cells were harvested with trypsin-EDTA and centrifuged. Then Cellswere then washed once with 500 ml of 1×PBS (0.14 mM NaCl, 2.7 mM KCl,7.9 μM Na2HPO4, 1.5 μM KH2PO4). 50 μl of PBS and 100 μl of SDS loadingbuffer (5% mercaptoethanol, 16 μM Bromphenolblue, 20 μM Xylene Cyanol,1.6 mM Ficoll 400) was added and cells were heated at +95° C. for 10minutes.

For the western blot analysis SDS-PAGE was prepared with 10% separationand 5% stacking gels in a SDS running buffer (25 mM Tris, 250 mM glysin,0.1% SDS). Cell samples and biotinylated molecular weigh marker wereloaded on the gel and electrophoresis was performed with 150 V for 1 h50 minutes. The transfer of proteins to a nitrocellulose membrane wasperformed at 100 V for 1.5 hours at room temperature with a cooler intransfer buffer.

The membrane was blocked in 5% milk in TBS (0.05 M Tris-Cl, 0.15 M NaCl,pH 7.5) for 30 minutes at room temperature. A primary antibody mixture,anti-AIRE6.1 (human) and anti-AIRE8.1 (mouse) antibodies at a dilutionof 1:100 in 5% milk in TBS, was added onto membrane and incubatedovernight at +4° C. The membrane was washed two times with 0.1% Tween inTBS for 5 minutes and once with TBS for 5 minutes. The secondaryantibody, biotinylated anti-mouse IgG at a dilution of 1:500 in 5% milkin TBS was incubated for 1 hour at room temperature. The membrane waswashed and horseradish peroxidase avidin D at a dilution of 1:1000 in 5%milk in TBS was added. The membrane was incubated at room temperaturefor 1 hour and washed. A substrate for the peroxidase was prepared of 5ml chloronaphtol, 20 ml TBS and 10 μl hydrogen peroxide and added ontomembrane. After the development of the color the membrane was washedwith TBS and dried.

The antibody detecting with human AIRE (anti-AIRE6.1) detected the AIREprotein expression in the preparates transfected with pS6 wthAIRE,pCAIRE and psiAIRE. The antibody detecting murine AIRE detected likewisethe murine AIRE in cells transfected with pS6 wtmAIRE and pCAIRE S1-4.The negative control (Super6wt) showed no AIRE/aire proteins.

6.10 Example 10 Detection of Cellular and Humoral Immune Response TowardHIV.1 Nef in Mice Immunized with the NNV-Nef Construct DNA Immunizations

To further study the induction of humoral immunity by the vectors of theinventions, 5-8 weeks old both male and female BALB/c (H-2d) mice wereused. For the DNA immunizations, the mice were anaesthetized with 1.2 mgof pentobarbital (i.p) and DNA was inoculated on shaved abdominal skinusing plasmid DNA coated gold particles. The inoculation was made withHelios Gene Gun (BioRad) using the pressure of 300 psi. The goldparticles were 1 μm in diameter, ˜1 μg of DNA/cartridge. The mice wereimmunized twice (on day 0 and day 7) with a total amount of DNA of 0.4or 8 μg/mouse. The control mice were immunized with 8 μg of the plainvector without the nef-gene, i.e. NNV-deltanef.

A blood sample was taken from the tail of the mice two weeks after thelast immunization. The mice were sacrificed four weeks after the lastimmunization and blood samples (100 μl) were collected to Eppendorftubes containing 10 μl of 0.5 M blotting (++vs. +) and in ELISA (higherOD, more mice in higher-dose above cut-off EDTA. The absolute number ofleukocytes/ml of blood was calculated from these samples for each mouse.The sera were collected for antibody assays and stored at −20° C. Thespleens were removed aseptically, weighted and then homogenized tosingle cell suspensions for use in T, B and NK cell assays and staining.

Detection of the Humoral Immunogenicity of the Vectors of the Invention

For the detection of Nef-specific antibodies by Western blotting, serumsamples from mice immunized with the vector constructs of the inventionwere diluted 1:100 to 5% milk in TBS and applied on nitrocellulosestrips made with recombinant HIV-1 Nef protein. For the preparation ofthe nitrocellulose strips, the purified recombinant protein was boiledin a sample buffer containing 1% SDS and 1% 2-mercaptoethanol, then runon a 10 or 12.5% polyacrylamide gel and subsequently transferred onto a0.45 μm nitrocellulose paper. The strips were first blocked with 2% BSAin 5% defatted milk-TBS and thereafter incubated with diluted sera(1:100) overnight. After incubation, unbound proteins were removed bywashing the strips three times with TBS-0.05% Tween-20 and twice withwater. After washings, the strips were probed with a 1:500 dilution ofbiotinylated anti-mouse IgG (Vector Laboratories, USA) for 2 hour. Afterfurther washings, horseradish peroxidase-avidin in a dilution of 1:1000(Vector Laboratories, USA) was added for 1 h, the strips were washedagain and the bound antibodies were detected with a hydrogen peroxidasesubstrate, 4-chloro-1-naphtol (Sigma, USA).

The sera were also tested in ELISA to determine the exact antibodytiters induced by each construct. Nef antibody ELISA was performed aspreviously described (Tähtinen et al., 2001). Briefly, Nunc Maxi Sorpplates were coated with 50 ng of Nef (isolate HAN), blocked with 2% BSAin phosphate buffered saline (PBS), and the sera in a dilution of 1:100to 1:25000 were added in duplicate wells for an overnight incubation.After extensive washings, the secondary antibody, peroxidase conjugatedanti-mouse IgG or IgM (DAKO), was added, and the plates were incubatedfor two hours and then washed. Color intensity produced from thesubstrate (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid, ABTS,Sigma) in a phosphate-citrate buffer was measured at 405 nm using aLabsystems Multiscan Plus ELISA-plate reader. The optical densitycut-off value for positive antibody reactions was determined as follows:

cut-off=OD(xl control mice sera)+3 SD.

Detection of the Cellular Immunogenicity of the Vectors of the Invention

To analyze the capacity of the vectors of the invention to inducecellular immunity, T-cell and B-cell assays as well as cell surfacestaining were performed.

T cell proliferation assay. The spleen cells were suspended to a finalconcentration of 1×106/ml RPMI-1640 (GibcoBRL) supplemented with 10% FCS(GibcoBRL), 1% penicillin-streptomycin (GibcoBRL) and 50 μMbetamercaptoethanol (Sigma). Cells were incubated in microtitre platesat 200 μl/well with media only or with different stimuli. The finalconcentrations of stimuli were: Con A 5 μg/ml, HIV-Nef-protein at aconcentration of 1 and 10 μg/ml, and a negative control antigen HIV-gagat a concentration of 1 and 10 μg/ml. All reactions were made inquadruplicates. On the sixth day of the incubation 100 μl of supernatantfrom each well was collected and stored at −80° C. for cytokine assays.Six hours before harvesting 1 μCi of 3H-thymidine (Amersham PharmaciaBiotech) was added to each well. The cells were harvested andradioactivity incorporated (cpm) was measured in a scintillationcounter. The stimulation indexes (SI) were calculated as follows

SI=mean experimental cpm/mean media cpm.

Lymphocyte activation. T and B cell activation was detected by doublesurface staining of fresh splenocytes with anti-CD3-FITC plusanti-CD69-PE (early activation marker) and anti-CD19-FITC plusanti-CD69-PE antibodies (all from Pharmingen). Stainings were analyzedwith flow cytometer (FACScan, Becton Dickinson).

CTL assays. Mouse splenocytes were co-cultured with fixed antigenpresenting cells (P-815 cells infected with MVA-HIV-nef or controlMVA-F6) for five days after which they were tested in a standard 4 hour51 chromium release assay [Hiserodt, J., et al., J Immunol 135 (1995)53-59; Lagranderie, M., et al., J Virol 71 (1997) 2303-2309) againstMVA-HIV-nef infected or control target cells. In CTL assays the specificlysis of 10% or more was considered positive.

Cytokine assay. IFN-gamma and IL-10 were measured fromantigen-stimulated cell culture supernatants in order to analyze,whether immunized mice develop a Th1 type or Th2 response. Thesupernatants were collected from antigen-stimulated cells as describedabove. Pro-inflammatory cytokines TNF-alfa and IL-10 were measured inthe sera of the immunized mice. All cytokines were measured withcommercial ELISA kits (Quantikine, R&D Systems).

Spontaneous proliferation. Spontaneous splenocyte proliferation wasdetected by 3H-thymidine uptake of the cells cultured in the medium onlyfor 6 days.

Anti-double strand (ds) DNA antibodies. dsDNA antibodies were measuredin the sera of immunized mice, positive control mice (mrl/lpr, agenerous gift from Dr. Gene Shearer, NIH, USA) and normal mice. Theantibodies were assayed with ELISA on poly-L-Lysine bounded lambda phagedsDNA. The results are shown in Tables 2 and 3.

Table 2 shows complete immunological results of the mice immunized withHIV-Nef plasmid DNA. Although HIV-1 Nef recombinant protein, which wasused for in vitro T cell stimulation, induced some non-HIV-specificproliferation of the cells in each immunized group, there was asignificant increase in the mean SI of mice immunized with 0.4 μg of theplasmid (mean SI=72.2) compared to others. Furthermore, negative controlprotein HIV-gag did not induce any T cell response. Only the T cells ofthe mice in the group that had nef-specific proliferation also producednef-specific IFN-gamma. None of the immunized mice had cells producingIL-10, which shows that the T cell response in the immunized mice was ofTh1 type and not of Th2 type. In contrast to the T cell response, miceimmunized with the higher concentration of nef plasmid DNA (8 μg) had astronger B cell response compared to mice immunized with 0.4 μg: thehumoral response in mice immunized with the higher dose was detectablealready three weeks after the last immunization and the responsedetected was stronger both in Western-). The antibodies detectedbelonged to IgG-class, no IgM response was detected. None of the micedeveloped E2 specific antibody.

The mice immunized with 0.4 μg of HIV-nef plasmid DNA had an increasednumber of leukocytes (6.38×106/ml) in the peripheral blood compared toother groups of immunized mice and normal mice (3.8×106/ml) (Table 3).The same mice had twice as much activated T cells (21%, CD3+CD69+)compared to other mice (9% and 10%). This finding is in correlation withthe positive T cell response to HIV-Nef (Table 2), since the mice with apositive T cell response to Nef also had an increased number ofactivated T cells in their spleens. The results of Table 3 also showthat none of the immunized mice developed anti-dsDNA anti-bodies ascompared to positive control sera (OD=1,208) indicating that there is noadverse effect of the immunization.

TABLE 2 HIV-1 HIV-1 HIV-1 nef gag IFN-g IL-10 nef E2 Mice SI* SI Th1 Th2Ab Ab NNV-Nef 8 1 6 1 − − ++ − 2 8 1 − − ++ − 3 13 2 − − ++ − 4 15 1 − −++ − 5 7 1 − − ++ − Mean 9.8 1.2 NNV-NEF 0.4 1 24 1 + − + − 2 112 1 +− + − 3 83 1 + − + − 4 73 1 + − + − 5 69 1 + − + − Mean 72.2 1 NNV-ΔNef8 1 6 1 − − − − 2 nt nt − − − − 3 11 1 − − − − 4 23 2 − − − − 5 12 1 − −− − Mean 13 1.25 SI* = stimulation index nt = not tested −′, negative+′, positive +′+′, strong positive

TABLE 3 CD3 CD3+CD69+ CD19 CD19+CD69+ anti-dsDNA % % % % ab Mice WBC ×10⁶/ml spleen spleen spleen spleen OD(1:10 dil) NM 1 0.355 2 0.255 30.231 Mean 0.280 NNV-Nef 8 1 5 nt nt nt nt 0.387 2 4.3 50 4 11 3 0.457 34.9 57 4 15 4 0.514 4 4.3 55 6 15 4 0.367 5 5.1 54 5 7 0 0.478 mean 4.7254 4.75 (9%) 12 2.75 0.441 NNV-Nef 0.4 1 3.9 nt nt nt nt 0.418 2 8 41 918 5 0.263 3 7.5 39 8 25 9 0.375 4 5 46 9 16 6 0.285 5 7.5 43 10 13 70.396 mean 6.38 42.25 9 (21%) 18 6.75 0.347 NNV-ΔNef 8 1 4.5 61 4 9 20.413 2 4.6 59 4 15 1 0.353 3 3.8 50 6 17 5 0.382 4 3.1 46 7 25 8 0.4485 3.5 nt nt nt nt 0.501 mean 3.9 54 5.25 (10%) 16.5 4 0.419 Normal mousemean WBC = 3.8 × 10⁶/ml nt, not tested a-dsDNA positive control sera ODwas 1.208 (1:10 dil)

6.11. Example 11 Safety and Immunogenicity of a Prototype HIV VaccineGTU-nef in HIV Infected Patients

Production of the NNV-2-Nef Vaccine (Check Whether NNV-2 or NNVwt-2 wasUsed)

The investigational vaccine NNV-2-Nef was prepared according to Example2 with the Manufacturing License No. LLDnro 756/30/2000 (issued by theFinnish National Agency for Medicines on Dec. 21, 2000).

The manufacturing processes performed fulfilled the current GoodManufacturing Practices (cGMP) requirements and provided plasmid DNApreparations suitable for use in clinical phase I and II studies. Themanufacturing process consisted of four steps:

a) Establishment of Master Cell Banks and Working Cell Banks

b) Fermentation

c) Purification

d) Aseptic filling of the vaccine

In detail, NNV-2-Nef was produced in E. coli bacteria. The Master CellBanks (MCBs) and Working Cell Banks (WCBs) containing E. coli DH5 alphaT1 phage resistant cell strain were established in accordance with thespecific Standard Operating Procedure from pure cultured and releasedResearch Cell Banks.

a) Establishment of Master Cell Banks and Working Cell Banks

The schematic procedure for establishing the cell bank system isillustrated below:

Thaw of one vial of Research Cell Bank [E. coli DH5 alpha T1 phageresistant cell strain (Gibco RBL) transformed with the NNV-2-Nefplasmid.

Inoculate of the culture on modified Luria Bertani medium plate(containing 25 μg/ml of kanamycin)

Incubate overnight (14-16 h) at 37° C.

Select of a single colony from the plate and inoculation into 50 ml ofmodified Luria Bertani medium (containing 25 μg/ml of kanamycin)

Incubate overnight (14-16 h) at 37° C.

Measure optical density of the bacterial culture (OD₆₀₀=2.0-6.0)

Add glycerol to bacterial culture

Divide the culture-glycerol mix to aliquots

Label and store the Master Cell Banks

Following the same diagram, the Working Cell Bank was established usingone vial of the Master Cell Bank as the starting material. The routinetests performed on the MCB and WCB were: microbiologicalcharacterization, absence of contamination, assessment of the plasmidstability by replica plating and the plasmid identity (restrictionenzyme digestion and sequencing).

b) Fermentation. In the fermentation the DH5 alpha T1 phage resistant E.coli strain (Gibco RBL, UK) transformed with NNV-2-Nef (WCB) was firstcultured on plate. From the plate a single colony was inoculated to a100 ml liquid pre-culture before the actual fermentation in thefermentation reactor. The fermentation was carried out in a 5 Ifermentor (B. Braun Medical) on a fed-batch system basis, after whichcells were harvested. The culture medium composition for one litrecontained 7 g of yeast extract, 8 g of peptone from soy meal, 10 g ofNaCl, 800 ml of water for injection (WFI), 1N NaOH, pH 7.0, kanamycin 50mg/ml (Sigma), silicon anti-foaming agent (Merck), 1M K₂PO₄(BioWhittaker).

In the beginning of the fermentation run, a 1 ml sample was takenthrough the harvesting tube to determine the initial cell density(OD₆₀₀). The pre-culture was used to inoculate the fermentation medium.During the fermentation, fresh culture medium and 1M potassium phosphatebuffer, pH 6.5-7.3, were fed to the reactor with the pumps. Addition ofthe medium allows replenishment of essential nutrients before they runout and phosphate buffer maintains the pH constant. When thefermentation process had continued for approximately 5 hours and at theend of the fermentation run (after approximately 10 hours offermentation), samples of 1 ml were taken as above and the cell densitywas measured. After the fermentation, the culture medium was centrifuged(10,000 rpm, 30 minutes, +4° C.) and the bacterial pellet (50-60 g) wasrecovered.

c) Purification. The methodology used for the purification of DNA wasbased on the QIAGEN process scale technology (Qiagen PlasmidPurification Handbook 11/98). The NN2-Nef was purified using thefollowing steps:

Resuspend the bacterial pellet in the resuspension buffer (100-150 ml,RT)

Lyse with the lysis buffer (100-150 ml, 5 minutes, RT)

Neutralize with the neutralization buffers (100-150 ml, +4° C.)

Incubate (30 minutes, +4° C.)

Centrifugate (10,000 rpm, 30 minutes, +4° C.)

Filtrate supernatant (0.22 micrometers)

Remove endotoxins with Endotoxin removal buffer (60-90 ml)

Equilibrate Ultrapure column with Equilibration buffer (350 ml, flowrate 10 ml/min)

Load lysate to the column (flow rate 4-6 ml/min)

Wash the column with Wash buffer (31, overnight, flow rate 4-6 ml/min)

Elute the plasmid DNA with Elution buffer (400 ml, flow rate 3.1 ml/min)

Filtrate the eluate (0.22 micrometer)

Precipitate DNA with isopropanol

Centrifuge (20000 g, 30 minutes, 4° C.)

Purified plasmid DNA

Buffers used within the purification were as follows. The resuspensionbuffer contained 50 mM Tris-Cl, pH 8.0, plus RNase A (50 mg); the lysisbuffer was 200 mM NaOH; the neutralization buffer was 3M potassiumacetate, pH 5.5; the endotoxin removal buffer contained 750 mM NaCl, 10%Triton X-100; 50 mM MOPS, pH7.0; the equilibration buffer contained 750mM NaCl, 50 mM MOPS, pH 7.0; the wash buffer contained 1 M NaCl, 50 mMMOPS, pH 7.0, 15% isopropanol; and elution buffer contained 1.6 M NaCl,50 mM MOPS, pH 7.0, 15% isopropanol.

d) Aseptic Filling

The purified DNA representing the final bulk was dissolved in 0.9%sterile physiological saline to a final concentration of 1 mg/ml andsterile filtered (0.22 micrometer) during the same day. The purifiedbulk was filled manually (filling volume 0.5 ml) in Schott Type 1 plusglass vials using a steam sterilized Finnpipette® and sterileendotoxin-free tips. The vials filled with the NN2-Nef vaccine wereclosed immediately, labelled and packed in accordance to the specificStandard Operating Procedure (SOP).

2. Administration of the Test Vaccine to the Patients

Ten HIV-1 infected patients undergoing Highly Active Anti-Retroviraltherapy (HAART) were immunized with the experimental DNA vaccineNN2-Nef, expressing the HIV-1 Nef gene (Clade B). For immunizations, twointramuscular injections in the gluteal muscle were given two weeksapart. The doses were 1 and 20 micrograms/injection. Blood samples weredrawn at −4, 0, 1, 2, 4, 8 and 12 weeks. The samples were analyzed forhumoral (ELISA, Western blot) and cell mediated immune response (T-cellsubsets, T-cell proliferation, ELISPOT, cytokine expression,intracellular cytokines).

A clinical examination was performed to each patient participating thestudy. The clinical examination included a patient interview (anamnesis)and weight determination. Cardiac and pulmonar functions were checked byauscultation and percussion, the blood pressure and heart rate wererecorded. Enlargement of lymph nodes, liver and thyroid gland weredetermined by palpation.

Laboratory tests to evaluate the safety of the vaccine were performed ateach visit. These tests included:

hematology: red blood cell count, haemoglobin, total and differentialWBC, platelet count, prothrombin time and activated partialthromboplastin time at baseline; mean erythrocyte corpuscular volume andhemoglobin content has been calculated.

Immunology: nuclear and ds-DNA antibodies.

Serum chemistries: total bilirubin, alkaline phosphatase, SGOT/SLT orSGPT/ALT, serum creatinine, protein electrophoresis, total serumcholesterol, triglycerides, glucose (at baseline), sodium, potassium,and calcium.

Urine analysis: dipstick protein, glucose, ketones, occult blood, bilepigments, pH, specific gravity and microscopic examination of urinarysediment (RBC, WBC, epithelial cells, bacteria, casts), when dipstickdetermination showed one or more abnormal values.

Viral load: Increases of more than one log 10 should be followed by aconfirmatory viral load estimate after two weeks.

None of the patients experienced subjective or objective adversereactions to the vaccination. No adverse laboratory abnormalities wereobserved in the panel of clinical chemistry tests (see material andmethods for details) performed repeatedly during the vaccination period.

The following immunological studies were performed:

Lymphocyte Proliferation Assay (LPA)

Peripheral blood mononuclear cells (PBMC) were isolated from heparinizedvenous blood by Ficoll-Hypaque density-gradient (Pharmacia)centrifugation and resuspended at 1×10⁶ cells/ml in RPMI 1640 medium(Gibco) supplemented with 5% pooled, heat-inactivated AB⁺ serum (Sigma),antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin; Gibco) andL-glutamine (complete medium, CM). Quadruplicate cultures were then setup in flat-bottomed micro titer plates (1×10⁵ PBMC/well) and the cellswere incubated for 6 days in the presence or absence of the followingstimuli: rNef (0.2, 1 and 5 μg/ml), GST (0.2, 1 and 5 μg/ml), purifiedprotein derivative of tuberculin (PPD, 12.5 μg/ml; StatensSeruminstitut), Candida albicans antigen (20 μg/ml; Greer Laboratories)and Phytohaemagglutinin (PHA; 5 μg/ml; Life Technologies). For the last6 h of the incubation period ³H-thymidine (1 μCi/well; Amersham) wasadded to the cultures and the cells were harvested onto glass fiberfilters and incorporated radioactivity was measured in a γ-counter.Results are expresses as delta cpm (cpm in the presence of antigen-cpmwithout antigen) or as stimulation index (cpm in the presence ofantigen/cpm without antigen).

The results are shown in FIGS. 26 and 27. None of the vaccines showedsignificant T-cell proliferative response to the test antigen, HIV-1 Nefbefore the vaccination. In contrast, 2 out of 5 vaccines in the groupthat had received 1 microgram dose of the test vaccine (patients 1 and3) (FIG. 26) and 2 out of 5 in the group receiving 20 micrograms of thetest vaccine (patients 9 and 10) (FIG. 27) showed a strong T-cellproliferative response after the first vaccination. After the secondvaccination, one (patient 2) vaccine responded in the 1-microgram group.

IFN-γ Assays

The type of immune response (Th1/Th2) induced by the vaccine wasevaluated by measuring interferon-gamma (IFN-γ) released in 6 days oldculture supernatant after antigen (rNef, rGST, PPD) or mitogen (PHA)stimulation of PBMC. For determinations, commercial ELISA kits (R&DQuantikine) were used. The assay employ the quantitative sandwich enzymeimmunoassay technique where a monoclonal antibody specific for IFN-γ hasbeen coated onto a microplate. Standards and samples are pipetted intothe wells and any IFN-γ present is bound by the immobilized antibody.After washing away any unbound substances, an enzyme-linked polyclonalantibody specific for IFN-γ is added to the wells. Following a wash toremove any unbound anti body-enzyme reagent, a substrate solution isadded to the wells and color develops in proportion to the amount of thecytokine bound in the initial step. The color development is stopped andthe intensity of the color is measured.

IFN-γ response data from patient# 1 is shown in FIG. 28. As can be seen,the vaccine responded to the rNef antigen by marked IFN-γ responsecorrelated with the T-cell proliferation, indicating that the responseseen in the vaccine is in fact of the Th1 type.

HIV-1 infection is characterized by low or totally lacking cell-mediatedimmune response towards all HIV proteins. The results show that it ispossible to induce a robust CMI in such patients with exceptionally lowdoses of the DNA vaccine NN2-Nef. The doses used were minimal to whathas generally been required with DNA vaccines. Thus, for instance, Merchannounced recently good results with their experimental HIV vaccine butthe doses required were from 1000 to 5000 micrograms (IAVI report,2002).

6.12 Example 12 Construction of the Plasmid Expressing Epstein-BarrVirus (EBV) EBNA-1 Protein and Containing 20 Binding Sites for EBNA-1(FR Element)

To construct a plasmid expressing Epstein-Barr virus (EBV) EBNA-1protein and containing 20 binding sites for EBNA-1 (FR element), BPV-1E2 binding sites were first replaced by EBV EBNA-1 binding sites (oriPwithout DS element). Plasmid FRE2d1EGFP (FIG. 29) was constructed byisolating the XmiI(AccI)/Eco32I (EcoRV) DNA fragment (blunt-ended withKlenow enzyme) of pEBO LPP plasmid (FIG. 29A) (the fragment contains 20binding sites for EBNA-1) and inserting it by blunt end ligation intothe SpeI/NheI site of s6E2d1EGFP (FIG. 29B) (blunt-ended with Klenowenzyme). The constructed plasmid FRE2d1EGFP (FIG. 29) was used as anegative control in further experiments. It contains binding sites forEBNA-1 protein instead of the BPV1 E2 10 binding sites, expressing E2,but not EBNA-1.

Next, the sequence encoding BPV-1 E2 protein in FRE2d1EGFP plasmid wasreplaced by a sequence encoding EBV EBNA-1 protein as follows. TheXmiI(AccI)/EcoRI fragment of pEBO LPP plasmid was isolated andblunt-ended with Klenow enzyme and inserted into the XbaI/XbaI site ofFRE2d1EGFP plasmid (blunted with Klenow enzyme). The vector FRE2d1EGFPwas previously grown in Escherichia coli strain DH5α (lacking Dam⁻methylation, because one XbaI site is sensitive for methylation. Theconstructed plasmid FREBNAd1EGFP (FIG. 30) expresses EBNA-1 protein andcontains 20 binding sites for EBNA-1.

For expression, Jurkat, human embryonic kidney cell line 293 (ATCC CRL1573) and mouse fibroblast cell line 3T6 cells (ATCC CCL 96) weremaintained in Iscove's modified Dulbecco's medium (IMDM) supplementedwith 10% fetal calf serum (FCS). Four million cells (Jurkat), 75%confluent dishes (293) or ¼ of 75% confluent dishes (3T6) were used foreach transfection, which were carried out by electroporation as follows.Cells were harvested by centrifugation (1000 rpm, 5 min, at 20° C.,Jouan CR 422), and resuspended in a complete medium containing 5 mMNa-BES buffer (pH 7.5). 250 μl cell of the cell suspension was mixedwith 50 μg of carrier DNA (salmon sperm DNA) and 1 μg (in the case ofJurkat and 3T6) or 5 μg (in the case of 293) of plasmid DNA andelectroporated at 200 V and 1000 μF for Jurkat cells, 170 V and 950 μFfor 293 cells and 230 V and 975 μF for 3T6 cells. The transfected Jurkatcells were plated on 6-cm dishes with 5 ml of medium; ⅓ of transfected293 and 3T6 cells were plated on a 6-cm dishes with 5 ml of medium and ⅔of the cells were plated on a 10-cm dishes with 10 ml of medium.

The transfected cells were analysed for the expression of d1EGFP protein(modified enhanced green fluorescent protein). All of the constructedplasmids expressed d1EGFP protein, which was detected by measuring thefluorescence using a flow cytometer. Because of the short half-life ofthe d1EGFP protein, it does not accumulate, and the expression of thisprotein reflects the presence of transcriptionally active plasmids inthe cells. Becton-Dickinson FACSCalibur system was used. The volume ofthe Jurkat cell suspension was measured before each time-point(approximately after every 24 hour) and if the volume was less than 5ml, the missing volume of medium was added. Depending on the cellsuspension density the appropriate volume was taken for measuring (1 or2 ml) and replaced with the same amount of medium. This was later takeninto consideration when the dilution was calculated.

For the first time-point, 293 cells from the 6-cm dish were suspended in5 ml of medium for measuring. In every following time-point half of thecells were taken from the 10-cm dish, suspended in 5 ml of medium andthen measured. An appropriate volume was added to the rest of the cellsuspension. For the first time-point, 3T6 cells from the 6-cm dish weresuspended in 1 ml of trypsine, which was then inactivated with 100 μl ofFCS. For every following time-point, cells from the 10-cm dish weresuspended in 2 ml of trypsin. 1 ml of this suspension was treated asdescribed previously. 9 ml of medium was added to the rest of thesuspension. The analyzed cells were taken out of the incubatorimmediately before the measurement. The appropriate flow speed (500-1000cells/sec) was determined before each time-point using cells transfectedonly with carrier DNA as a control. Three different parameters were usedto measure size, surface structure and fluorescence of the cells.

The results are presented as graphs in FIG. 31. Cells transfected onlywith carrier DNA were used to measure the auto-fluorescence of thecell-line. 1% of this auto-fluorescence was considered as backgroundfluorescence and was subtracted later from the d1EGFP fluorescence. Thereceived data was analyzed using Microsoft Excel program.

Percentages of the d1EGFP expressing cells were calculated using cellstransfected with the carrier only as a negative control for backgroundfluorescence. As shown in FIG. 33, the two vectors were maintained inthe cells with different kinetics.

The number of the d1EGFP expressing cells was calculated taking thedilutions into consideration using cells transfected with the carrieronly as a negative control for background fluorescence. As seen fromFIG. 53, the plasmids expressing EBNA-1 and carrying EBNA-1 specificmultimeric binding sites are maintained very efficiently in thetransfected cells. At day 1 after transfection approximately 8×10⁴ cellsexpressed EGFP. At day 8, in the case of maintenance vector(FREBNAd1EGFP), the number of the plasmid positive d1EGFP expressingcells had increased ten times to 8×10⁵. With the plasmid lacking EBNA-1expression (FRE2d1EGFP) or having no EBNA-1 binding sites, the number ofplasmid positive cells was retained or in many cases decreased. Thisfact reflects the mechanism for segregation/partitioning Epstein-Barrvirus. Maintenance and segregation function by EBNA1 and EBNA-1 bindingsites provides maintenance function to the plasmid if EBNA-1 isexpressed and plasmid carries EBNA-1 binding sites. The same mechanismand the same components actually provide the segregation function toEpstein-Barr Virus in the latent phase of life-cycle.

Similar results were obtained also in human embryonic cell line 293 andmouse cell line 3T6 (FIG. 34). As a control for the maintenance for 293and 3T6 cells, s6HPV11 and 2wtFS, respectively, were used.

6.53 Example 13 The Immunogenicity of GTU-Multigene Vectors

The Immunogenicity of GTU-1-Multigene Vectors

The immunogenicity of six different multi-gene vaccine constructsprepared in Example 12, i.e. GTU-1-RNT, GTU-1-TRN, GTU-1-RNT-CTL,GTU-1-TRN-CTL, GTU-1-TRN-optgag-CTL, and GTU-1-TRN-CTL-optgag vectorswere tested in mice. The vectors were transformed into TOP10 or DH5alphacells, and the MegaPreps were prepared using commercial Qiagen columns.Endotoxins were removed with Pierce Endotoxin Removal Gel.

The test articles were coated on 1 μm gold particles according to theinstructions given by the manufacturer (Bio-Rad) with slightmodifications. Balb/c mice were immunized with a Helios Gene Gun using apressure of 400 psi and 0.5 mg gold/cartridge. Mice were immunized threetimes at weeks 0, 1, and 3. Mice were sacrificed two weeks after thelast immunization.

Mice were divided into six test groups (5 mice/group), which received3×1 μg DNA as follows:

Group 1. GTU-1-RNT

Group 2. GTU-1-TRN

Group 3. GTU-1-RNT-CTL

Group 4. GTU-1-TRN-CTL

Group 5. GTU-1-TRN-optgag-CTL

Group 6. GTU-1-TRN-CTL-optgag

Group 7. Control mice immunized with empty gold particles not loadedwith DNA.

The humoral response was followed from tail-blood samples from eachmouse. First pre-immunization sample was taken from anesthetized micebefore the first immunization was given. Second sample was taken fromanesthetized mice before the third immunization. At sacrifice, wholeblood sample was used for white blood cell counting, and serum wascollected for humoral immunity tests.

The blood samples were tested for antibodies with ELISA using a standardprocedure. Nunc Maxi Sorp plates were coated with 100 ng of Nef, Rev,Tat, Gag, CTL or E2 proteins, blocked and sera at a dilution of 1:100were added in duplicate wells for an overnight incubation. Afterwashing, the plates were incubated for 2 hours with a diluted (1:500)secondary antibody, peroxidase conjugated anti-mouse IgG (DAKO). Colorintensity produced from the substrate(2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) inphosphate-citrate buffer was measured at 405 nm using LabsystemsELISA-plate reader.

All vectors induced Nef antibodies in all mice, whereas none of the miceshowed E2, CTL or Rev antibodies (FIGS. 35, 36, and Table 4) Some of themice immunized with GTU-1-RNT or GTU-1-RNT-CTL also developed Tatanti-bodies (FIG. 36 and Table 4). Furthermore, mice immunized withvectors containing the optgag sequence developed also Gag antibodies,but the construct GTU-1-TRN-optgag-CTL was a better antibody inducerthat the construct GTU-1-TRN-CTL-optgag (FIG. 37 and Table 4). Theantibodies induced were mainly of the IgG1 class indicating a Th2 typeof response usually seen with gene gun immunization. The antibody assaysshown below were done from the sera collected when mice were sacrificed.

The results show that a multigene construct, expressing several HIVgenes as a fusion protein, can induce an immune response to most of thegene products. The orientation and order of the genes in the multigeneand corresponding proteins in the fusion proteins affects the results,however, dramatically. Thus, a response against Tat was seen only whenthe Tat gene was placed inside the fusion protein (vectors with RNTmotif) and not when Tat was the amino terminal protein (vectors with theTRN motif). Response to the Gag proteins was seen only with the vector,where Gag was placed before the CTL containing a stretch of Th and CTLepitopes.

TABLE 4 Immuno III A mice ELISA results (OD mean of five mice) GroupImmunogen number Nef (own prot) Tat Rev Gag CTL GTU-1-RNT 1 2.194 1.3910.31 0.155 0.36 GTU-1-TRN 2 1.849 0.197 0.252 0.302 0.38 GTU-1-RNT- 31.922 0.555 0.295 0.154 0.439 CTL GTU-1-TRN- 4 1.677 0.211 0.298 0.140.425 CTL GTU-1-TRN- 5 1.722 0.182 0.24 0.667 0.381 optgag-CTLGTU-1-TRN- 6 0.547 0.225 0.322 0.228 0.43 CTL-optgag Controls 7 0.3160.226 0.282 0.16 0.405 Percent of Nef Tat Rev Gag CTL Immunogen Groupresponse response response response response GTU-1-RNT 1 100 80 0 0 0GTU-1-TRN 2 100 0 0 20 0 GTU-1-RNT- 3 100 40 0 0 0 CTL GTU-1-TRN- 4 1000 0 0 0 CTL GTU-1-TRN- 5 80 0 0 60 0 optgag-CTL GTU-1-TRN- 6 100 0 0 200 CTL-optgag

6.14, Example 14 Expression of Hybrid Protein Expressing Nef, Rev andTat in Different Combinations (Multireg)

For the production of HIV multi-gene vectors, GTU-1 vector with amulti-cloning site (FIG. 38A) was used as a backbone. Intact Nef, Revand Tat coding sequences were amplified by the polymerase chain reaction(PCR) and attached to each other in various orders to multi-regulatory(multireg) antigen coding reading frames (Nef-Tat-Rev, Tat-Rev-Nef,Rev-Tat-Nef, Tat-Nef-Rev and Rev-Nef-Tat; Sequences Id. No. 1 to 5,respectively). These sequences were cloned to the Bsp191 and NotI sitesof the GTU-1 vector.

Similarly, Nef protein expressing GTU-2 and GTU-3 vectors (FIGS. 38B and38C; see also FIG. 6B for NNV-2wt)) were also used as backbones for theproduction of HIV multigene vectors. Additionally, the vector super6wtexpressing destabilized enhanced green fluorescent protein or d1EGFP(super6wtd1EGFP; FIG. 17 and FIG. 38D) and plasmid utilizing the EBNA-1protein and its binding sites (FREBNAd1EGFP; FIG. 38E) were used as aGene Transfer Unit (GTU) platform. For control “non-GTU” vectors, aregular cytomegalovirus (CMV) vector NNV-Rev expressing Rev and aplasmid EBNA-1 and E2BS containing d1EGFP plasmid (NNV-Rev andE2BSEBNAd1EGFP, respectively; FIGS. 38G and F) were used as backbones.

For the preparation of different GTU-2 and GTU-3 vectors (pNRT, pTRN,pRTN, pTNR and pRNT; and p2TRN and p2RNT; and p3RNT, FIGS. 39A-E, 39F-Gand 39H, respectively), the Nef gene in vectors GTU-2Nef and GTU-3Nefwas substituted by the respective multireg antigen using NdeI and Pag Isites. The sequence of the letters N(ef), R(ev) and T(at) in the nameshows the position of respective coding sequences of the protein in themulti-gene. Also two vectors, which contain the IRES element placed intothe SalI sites following either the multi-antigen or E2 codingsequences, were prepared (pTRN-iE2-GMCSF and pTRN-iMG-GMCSF,respectively; FIGS. 39I and J). The latter sequence, which controls thetranslation of the coding sequence of the mouse granulocyte-magrophagecolony stimulating factor (GM-CSF), was cloned into the single BspTIsite introduced with IRES.

Additionally, a set of the vectors, in which only immunodominant partsof the regulatory proteins were used for building up the polyproteins,were cloned into the Bsp119I and NotI sites of the GTU-1 (pMV1 NTR,pMV2NTR, pMV1N11TR and pMV2N11TR; FIGS. 40A-D). In case of the pMV2constructs, linkers that could be digested by intracellular proteasesseparate the regions of the multi-antigene derived from differentregulatory proteins.

Further, GTU-1, GTU-2 and GTU-3 vectors, which express the structuralproteins encoded by the gag gene or an artificial polyprotein composedby previously described CTL epitopes, were prepared. The codingsequences were cloned as Bsp119I and Not I digested PCR fragments intothe GTU-1 vector (pCTL=BNmCTL, pdgag=pBNdgag, psynp17/24=pBNsynp17+24,poptp17/24=pBNoptp17/24; FIGS. 41A-D), and transferred in a Nde I-Pac Ifragment to the GTU-2 (p2mCTL and p2optp17/24; FIGS. 41E and F) andGTU-3 (p3mCTL and p3optp17/24; FIGS. 41G and H).

The coding segment designated as CTL (Sequence Id. No. 10) containsfragments from pol and env regions involving many previously identifiedCTL epitopes. The codon usage is optimized so that only codons usedfrequently in human cells are involved. This coding sequence alsocontains a well-characterized mouse CTL epitope used in potency assayand an epitope for recognition by anti-mouse CD43 antibody. Also, adominant SIV p27 epitope was included for use in potency studies inmacaques.

The dgag contains truncated p17 (start at 13 aa) +p24+p2+p7 (p1 and p6are excluded) (Sequence Id. No. 11) of gag region of the Han2 isolate.The synp17/24 (Sequence Id. No 12) codes for the p17+p24 polypeptide ofthe Han2 HIV-1. The codon usage is modified to be optimal in humancells. Also, previously identified AU rich RNA instability elements wereremoved by this way. The optp17/24 coding (Sequence Id. No. 13) regionis very similar to the synp17/24 with the exception that the twosynonymous mutations made therein do not change the protein compositionbut remove a potential splicing donor site.

Further, a set of the multi-HIV vectors, which contain both a multiregantigen and structural antigens as a single polyprotein, were created:pTRN-CTL, pRNT-CTL, pTRN-dgag, pTRN-CTL-dgag, pRNT-CTL-dgag,pTRN-dgag-CTL, pRNT-dgag-CTL, pTRN-optp17/24-CTL, pTRN-CTL-optp17/24,and pRNT-CTL-optp17/24; p2TRN-optp17/24-CTL, p2RNT-optp17/24-CTL,p2TRN-CTL-optp17/24, p2RNT-CTL-optp17/24,p2TRN-CTL-optp17/24-iE2-mGMCSF, and p2RNT-CTL-optp17/24-iE2-mGMCSF; andp3TRN-CTL-optp17/24, p3RNT-CTL-optp17/24, p3TRN-CTL-optp17/24-iE2-mGMCSF, and p3RNT-CTL-optp17/24-iE2-mGMCSF, FIGS. 42A-T.

For cloning, as a first step the STOP codon was removed from theregulatory multi-antigen coding sequences. Then the structural antigencoding sequences were added by cloning into the NotI site at the end ofthe frame so that a NotI site was reconstituted. If both CTL and gagwere added, the first antigen coding sequence was without the STOPcodon. Generally, the clonings were made in context of GTU-1 and formaking the respective GTU-2 (p2 . . . ) and GTU-3 (p3 . . . ) vectors,the Nef gene in the plasmids GTU-2Nef and GTU-2Nef was replaced usingsites for NdeI and Pag I. However, the RNT-optp17/24-CTL antigen wasbuilt up directly in GTU-2 vector.

The HIV multi-antigen was cloned to the vectors super6wtd1EGFP andFREBNAd1EGFP instead of the d1EGFP using sites for Eco105I and NotI(super6 wt-RNT-CTL-optp17/24 and FREBNA-RNT-CTL-optp17/24; FIGS. 43V and42 U, respectively). If indicated, the IRES and mouse mGM-CSF werecloned into the GTU-2 and GTU-3 vectors behind the E2 coding sequenceinto the sites Mph1103I and Eco91I from pTRN-iE2-mGMCSF (cut out usingsame restrictases).

Finally, “non-GTU” control vector E2BSEBNA-RNT-CTL-optp17/24 (FIG. 42W)for the system utilizing EBNA-1 (contains EBNA-1 expression cassettewith E2 binding sites) was made in a similar way as theFREBNA-RNT-CTL-optp17/24. The regular CMV vector pCMV-RNT-CTL-optp17/24expressing the multi HIV antigen (FIG. 42D) was made by cloning themulti-HIV coding fragment from respective GTU-1 vector using sites forNdeI and Pag I.

6.5.2 Expression Properties of the Multireg Antigens Carrying OnlyImmunodominant Regions of the Regulatory Proteins.

1. Intracellular Localization of the MultiREG Antigens

The intracellular localization of MultiREG antigens expressed by thevectors of the invention was studied by in situ immunofluorescence in RDcells using monoclonal antibodies against Nef, Rev and Tat proteinsessentially as described in Example 4. The results are summarized inTable 5 and illustrated in FIG. 45. All antigens that are comprised ofintact Nef, Rev and Tat proteins showed exclusive localization incytoplasm. The aberrant protein initially designed as N(ef)T(at)R(ev),which has a frame-shift before the Rev sequence, showed only the nuclearlocalization. MultiREG antigens carrying truncated sequences of theregulatory proteins were localized in cytoplasm. In this cases distinctstructures like “inclusion bodies” were frequently observed. The samewas true for antigens, which carried the protease sites expressed frompMV2 vectors. However in these cases the proteins in nucleus were alsodetected (FIG. 45).

TABLE 5 Intracellular localization in multireg antigenes Constructanti-Nef anti-Rev anti-Tat empty negative negative negative GTU-1 pTRNstrong staining in cytoplasm good staining in cytoplasm positivestaining in cytoplasm pNTR strong staining in negative positive stainingin nucleus, nucleolus nucleus pRNT strong staining in cytoplasm goodstaining in cytoplasm good staining in cytoplasm pNRT strong,cytoplasmic good staining in cytoplasm good staining in cytoplasm pRTNstrong, cytoplasmic good staining in cytoplasm positive staining incytoplasm pTNR strong, cytoplasmic good staining in cytoplasm goodstaining in cytoplasm pMV1NTR strong, cytoplasmic cytoplasmic +inclusions cytoplasmic + inclusions pMV1N11TR strong cytoplasmic +inclusions cytoplasmic + inclusions cytoplasmic + inciusions pMV2NTRinclusions in nuclei and inclusions in nuclei inclusions in nuclei incytopi. and cytoplasm and cytoplasm pMV2N11TR only inclusions, in nucleionly inclusions in only inclusions in and in cytopi nuclei and in cytopinuclei and in cytopi.

The intracellular localization of dgag and p17+p24 proteins was alsoanalyzed in RD cells by immunofluorescence with monoclonal anti p24antibodies. In accordance with the Western blot results in Jurkat cells,the dgag could not be detected. However, the p17/24 protein showedlocalization in plasma membranes (FIG. 45). The localization of CTLprotein was not analyzed, because no suitable antibody was available.

6.6 Example 15 Analysis of Vectors Encoding Recombinant GAG Antigens andCytotoxic T-Cell Epitopes (CTL) from POL

6.6.1. Expression

Analysis of expression of the vectors expressing CTL cds or proteinsfrom the gag region were performed by western blot. As seen on FIGS. 46Aand 46B, the CTL and dgag expression was clearly demonstrated in Cos-7cells as predicted size proteins (25 kD and 47 kD, respectively). Theco-transfection of the Nef, Rev and Tat significantly enhanced theexpression of the dgag protein. We interpret this as a result of REVprotein action on the GAG mRNA expression We also tried to express thedgag protein from GTU-1 vector in Jurkat cells, but we failed to detectany signal (FIG. 46C). The analysis of the codon usage showed that wtGAG sequence had not optimal codon usage for human cells. When the codonusage was optimized (constructs psynp17/24 and poptp17/24), strongp17+p24 (40 kD) protein expression was detected in Jurkat cells (FIGS.46C and 46D).

6.6.2. Intracellular Localization

For dgag and p17+p24 proteins, the intracellular localization was alsoanalyzed in RD cells by immunofluorescence with anti p24 Mab. Similar tothe western blot results in Jurkat cells, the dgag could not bedetected. The p17/24 protein showed localization in plasma membranes(FIG. 47). The localization of CTL protein was not analyzed caused bylacking of suitable antibody

6.7 Example 16 Multireg+Structural Proteins as Multi-HIV AntigenExpression

As next step, the expression of the Multi HIV antigenes consisting ofboth, regular multigene together with gag encoded protein and/or CTLmultiepitope as single polypeptide was analysed. On FIG. 48, the Westernblot shows the expression of several multiHIV-antigenes expressingvectors transfected to the Cos-7 cells. It is clearly seen that theexpression levels of all regulatory+structural multi-antigenes aresignificantly lower than of the RNT or TRN proteins. All tested MultiHIVantigenes migrate in the gel as distinct bands near the position ofpredicted size (73 kD for multireg+CTL; 95 kD for multireg+dgag and 120kD for multireg+CTL+dgag). Similar to the RNT and TRN, the RNT-CTLmigrates more slowly than TRN-CTL. Also, in cases of both TRN and RNTconstructs, the MultiREG-CTL-dgag combination showed higher expressionlevel than MultiREG-dgag-CTL.

More detailed analysis of the multiHIV antigenes was performed in Jurkatcells. For this reason, most of the constructed MultiHIV antigenes(multireg+structural), included the MultiREG+CTL+optp17/24 (withpredicted size 113 kD) were analyzed by Western blotting usingantibodies against different parts of the antigene. The results arepresented on FIG. 49 are principally similar to those were reported inprevious section in case of Cos-7 cells. As it was seen in the previousexperiments, the dgag containing multi-antigenes express very low levelsof the hybrid protein in Jurkat cells. The expression from the vectorpTRNdgag was undetectable on all blots. In lanes loaded material fromcells transfected with other dgag containing antigene expressionvectors, very faint signals only on the Nef Mab hybridized blot weredetected at positions of predicted sizes. In contrast, if the dgag partis replaced with the codon optimized p17/24, the expression levelincrease was observed. Because the TRN-CTL-optp17/24 and RNT-optp17/24were initially chosen for further analysis, the expression of theantigenes was analyzed from all GTU vectors containing these expressioncassettes. Also, the E2 protein expression from these plasmids wasanalyzed. The results are illustrated on FIG. 50. There are no bigdifferences between the vectors in expression levels of bothmulti-antigene and the E2 protein. The E2 expression level is notsignificantly influenced by presence of IRES element followed mouseGM-CSF gene in the plasmid, translated from the same mRNA as the E2.

6.7.1. Example 17 Maintenance of Expression of Antigen

The maintenance of the plasmid in a population of dividing cells wasproved using the green fluorescent protein and Nef protein as markers.The maintenance of the expression of the RNT-CTL-optp17/24 antigenproduced from different GTU or non-GTU vectors was also analyzed.Specifically, GTU-1 (p RNT-CTL-optp17/24), GTU-2 (p2 RNT-CTL-optp17/24),GTU-3 (p3 RNT-CTL-optp17/24), super6wt (super6wt-RNT-CTL-optp17/24)vectors each utilize the E2 protein and its binding sites for theplasmid maintenance activity. In this experiment, also EBNA-1 and itsbinding site utilizing GTU vector FREBNA-RNT-CTL-optp17/24 was included.As negative controls, “non-GTU” plasmid containing a mixed pair of theEBNA-1 expression cassette together with E2 binding sites(E2BSEBNA-RNT-CTL-optp17/24) was used. Also, regular CMV expressionvector pCMV-RNT-CTL-optp17/24 was used.

Jurkat cells were transfected with equimolar amounts of the plasmids andthe antigen expression was studied at 2 and 5 days post-transfectionusing a monoclonal anti-Nef antibodies. Transfection with carrier DNAonly was used as a negative control. The results are presented in FIG.51.

As it seen from FIG. 51, the expression is detectable only from GTUvectors at the second time-point. The antigen expression from theFREBNA-RNT-CTL-optp17/24 was lower at both time-points, because, unlikeE2, the EBNA-1 does not have transcription activation ability.

Also the intracellular localization of the multireg+structuralpolyproteins was studied by in situ immunofluorescence analysis in RDcells essentially as described in Example 4. The results are presentedin FIG. 52.

In all cases localization only in cytoplasm was detected using eithermonoclonal anti-Nef or anti-p24 antibodies. In accordance with Westernblot data, the expression level of optp17/24 containing proteins wasmuch stronger than dgag fragment containing antigens.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims along with the full scope ofequivalents to which such claims are entitled.

1. A method for treating an HIV disease in a subject in need of saidtreatment, said method comprising: administering to said subject atherapeutically effective amount of a DNA vaccine comprising anexpression vector and a pharmaceutically acceptable excipient, whereinsaid expression vector comprises: (a) a heterologous promoteroperatively linked to a DNA sequence encoding a nuclear-anchoringprotein, wherein said nuclear-anchoring protein comprises: (i) a DNAbinding domain which binds to a specific DNA binding sequence, and (ii)a functional domain of the Bovine Papilloma Virus Type 1 E2 protein,wherein said functional domain binds to a nuclear component; (b) amultimerized DNA sequence that forms a binding site for said nuclearanchoring protein; and (c) at least one expression cassette comprising aDNA sequence encoding a protein or peptide that stimulates an immuneresponse specific to the protein or peptide; wherein said expressionvector lacks an origin of replication functional in mammalian cells. 2.The method of claim 1, wherein said nuclear component is selected fromthe group consisting of mitotic chromatin, the nuclear matrix, nucleardomain 10 (ND10), and nuclear domain PML oncogenic domain (POD).
 3. Themethod of claim 1, wherein said nuclear-anchoring protein is achromatin-anchoring protein, and said functional domain binds mitoticchromatin.
 4. The method of claim 1, wherein said nuclear-anchoringprotein comprises a hinge or linker region.
 5. The method of claim 1,wherein said nuclear-anchoring protein is a natural protein of viralorigin.
 6. The method of claim 1, wherein said nuclear-anchoring proteinis an artificial protein.
 7. The method of claim 1, wherein saidexpression cassette comprises a DNA sequence of HIV origin.
 8. Themethod of claim 7, wherein said DNA sequence of HIV origin encodes anon-structural regulatory protein of HIV, or an immunogenic fragmentthereof.
 9. The method of claim 8, wherein said nonstructural regulatoryprotein of HIV is selected from the group consisting of Nef, Tat andRev.
 10. The method of claim 9, wherein said nonstructural regulatoryprotein of HIV is Nef.
 11. The method of claim 7, wherein said DNAsequence of HIV origin encodes a structural protein of HIV, or animmunogenic fragment thereof.
 12. The method of claim 11, wherein saidDNA sequence of HIV origin is HIV gp120/gp160.
 13. The method of claim1, wherein said vector comprises: (a) a first expression cassettecomprising a DNA sequence encoding Nef, Tat or Rev; and (b) a secondexpression cassette comprising a DNA sequence encoding Nef, Tat or Rev.14. The method of claim 1, wherein said vector comprises: (a) a firstexpression cassette comprising a DNA sequence encoding Nef, Tat or Rev;and (b) a second expression cassette comprising a DNA sequence encodinga structural protein of HIV.
 15. The method of claim 1, wherein: saidDNA binding domain comprises the DNA binding domain of the BovinePapilloma Virus Type 1 E2 protein; and said multimerized DNA sequencecomprises multimerized E2 binding sites.
 16. The method of claim 1,wherein: said nuclear-anchoring protein comprises the Bovine PapillomaVirus Type 1 E2 protein, and said multimerized DNA sequence comprisesmultimerized E2 binding sites.
 17. The method of claim 1, wherein saidexpression cassette comprises a DNA sequence encoding a fusion proteincomprising the following components: (A) Rev, Nef, Tat (RNT); (B) opt17/24; and (C) Cytotoxic T cell epitopes (CTL).
 18. The method of claim17, wherein the order of the components from the 5′ end to the 3′ end ofsaid fusion protein is A+B+C.
 19. The method of claim 17, wherein thecomponents A, B, and C comprise the sequences of SEQ ID NOS: 5, 13 and10, respectively.
 20. A DNA vaccine comprising an expression vector anda pharmaceutically acceptable excipient, wherein said expression vectorcomprises: (a) a heterologous promoter operatively linked to a DNAsequence encoding a nuclear-anchoring protein, wherein saidnuclear-anchoring protein comprises: (i) a DNA binding domain whichbinds to a specific DNA binding sequence, and (ii) a functional domainof the Bovine Papilloma Virus Type 1 E2 protein, wherein said functionaldomain binds to a nuclear component; (b) a multimerized DNA sequencethat forms a binding site for said nuclear anchoring protein; and (c) atleast one expression cassette comprising a DNA sequence encoding aprotein or peptide that stimulates an immune response specific to theprotein or peptide; wherein said expression vector lacks an origin ofreplication functional in mammalian cells; and wherein said expressioncassette comprises a DNA sequence encoding a fusion protein comprisingthe following components: (A) Rev, Nef, Tat (RNT); (B) opt 17/24; and(C) Cytotoxic T cell epitopes (CTL).
 21. The DNA vaccine of claim 20,wherein the order of the components from the 5′ end to the 3′ end ofsaid fusion protein is A+B+C.
 22. The DNA vaccine of claim 20, whereinthe components A, B, and C comprise the sequences of SEQ ID NOS: 5, 13and 10, respectively.
 23. The DNA vaccine of claim 21, wherein thecomponents A, B, and C comprise the sequences of SEQ ID NOS: 5, 13 and10, respectively.